Lysin polypeptides active against Gram-negative bacteria

ABSTRACT

The present disclosure provides methods and compositions useful for the prophylactic and therapeutic amelioration and treatment of infections caused by Gram-negative bacteria, including Pseudomonas aeruginosa. The disclosure further provides compositions and methods of incorporating and utilizing lysin polypeptides of the present disclosure for augmenting the efficacy of antibiotics generally suitable for the treatment of Gram-negative bacterial infection.

STATEMENT OR RELATED APPLICATIONS

This patent application claims the priority of U.S. Provisional PatentApplication 62/220,212 filed Sep. 17, 2015, and U.S. Provisional PatentApplication 62/247,619 filed Oct. 28, 2015; the contents of theseprovisional applications are hereby incorporated by reference in theirentirety.

BACKGROUND OF THE DISCLOSURE Technical Field

The present disclosure relates generally to the prophylaxis andtreatment of infections caused by the Gram-negative bacteria. Morespecifically, the disclosure relates to agents and compositions capableof preventing and/or inhibiting growth of Gram-negative bacteria.

Description of the Related Art

Gram-negative pathogens pose a significant threat with evolvingresistance to nearly all drugs previously considered for treatment. Ofparticular concern are healthcare-associated infections involving theGram-negative pathogen Pseudomoms aeruginosa which can developresistance to numerous antimicrobial agents such as antibiotics,including but not limited to ciprofloxacin, levofloxacin, gentamicin,cefepime, imipenem, meropenem (Lister et al. Clin Microbiol Rev. 4:582-610 (2009)). In addition to resistance to individual drugs, theemerging and increasing prevalence of multidrug-resistant strainscombined with the paucity of new antibiotics is cause for alarm. Noveland effective treatments for Gram-negative infections are clearly neededto address the threat from multi-drug-resistance. One very promisingapproach is based on the use of bacterial peptidoglycan hydrolases, orPGHs, including lysins, autolysins, and some bacteriocins to degrade amajor structural component of the bacterial cell wall (i.e.,peptidoglycan). PGHs include glucosaminidases and muramidases (i.e.,lysozymes), which cleave the sugar backbone of peptidoglycan,endopeptidases, which cleave the stem-peptide or cross-bridge, orL-alanine amidases, which cleave the amide bond connecting the sugar andpeptide moieties (Bush K., Rec Sci Tech. (1):43-56 (2012); Reith J. etal. Appl Microbiol Biotechnol. (1):1-11 (2011)).

Work over the past 14 years has shown that PGHs can be recombinantlyexpressed, purified, and added exogenously to sensitive bacteria forrapid bacteriolysis. This “lysis from without” phenomenon is the basisof an effective antibacterial strategy currently under development forseveral Gram-positive bacterial pathogens. However, compared toGram-positive bacteria, the use of lysins for treatment of Gram-negativebacterial infections has been limited due to the existence of theadditional membrane layer within the bacterial cell wall. Thisadditional layer, known as the outer membrane (OM), hinders the accessof lysins to their peptidoglycan substrates in the cell wall.Nonetheless, recently, several PGHs from Gram-negative bacteria andassociated bacteriophages have been reported with some innate ability tokill Gram-negative bacteria (Lood et al., Antimicrob Agents Chemother,4:1983-91, (2015)). For bactericidal Gram-negative lysins, the activitymay be dependent on positively charged (and amphipathic) N- andC-terminal alpha helical domains in the native sequences, which enablebinding to the anionic OM and effect translocation into the subjacentpeptidoglycan (Lai et al. Microbiol Biotechnol, 90:529-539 (2011)).Recently, researchers have used this knowledge to create “artilysins,”engineered lysins with added cationic peptides for antibacterialactivity against Gram-negative Pseudomonas aeruginosa and Acinetobacterbaumannii (Briers et al., Antimicrob Agents Chemother. 58(7): 3774-84(2014)). These artilysins consist of positively charged PGHs (activeagainst Gram-positive bacteria) fused to exogenously-derived cationicpeptides that are not related to or derived from lysins (Briers et al.,Antimicrob Agents Chemother. 58(7): 3774-84 (2014); Briers et al. MBio.4:e01379-14 (2014); U.S. Pat. No. 8,846,865).

The citation of references herein shall not be construed as an admissionthat such references are relevant or that they constitute prior art tothe present disclosure.

SUMMARY OF THE DISCLOSURE

In one aspect, the present disclosure provides a pharmaceuticalcomposition comprising an effective amount of an isolated lysinpolypeptide comprising an amino acid sequence having at least 80% or atleast 85% or at least 90% or at least 95% identity to a polypeptidesequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO:10, or a fragment thereof having lysin activity, wherein the lysinpolypeptide inhibits the growth, or reduces the population, or kills P.aeruginosa and optionally at least one other species of Gram-negativebacteria; and a pharmaceutically acceptable carrier.

In some embodiments, lysin polypeptide or fragment is present in anamount effective to inhibit the growth, to reduce the population, or tokill P. aeruginosa and optionally at least one other species ofGram-negative bacteria.

In some embodiments, the pharmaceutical composition is a solution, asuspension, an emulsion, an inhalable powder, an aerosol, or a spray.

In some embodiments, the pharmaceutical composition further comprisesone or more antibiotics suitable for the treatment of Gram-negativebacteria.

In another aspect, the disclosure provides a vector comprising anisolated polynucleotide comprising a nucleic acid molecule that encodesa lysin polypeptide comprising an amino acid sequence having at least80% or at least 85% or at least 90% or at least 95% sequence identity toa sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO:10, or a fragment thereof having lysin activity, wherein the encodedlysin polypeptide inhibits the growth, or reduces the population, orkills P. aeruginosa and optionally at least one other species ofGram-negative bacteria or a complementary sequence of saidpolynucleotide.

In another aspect, the disclosure provides a recombinant expressionvector comprising a nucleic acid encoding a lysin polypeptide comprisingan amino acid sequence at least 80% or at least 85% or at least 90% orat least 95% identical to a sequence selected from the group consistingof SEQ ID NO: 1-SEQ ID NO: 10, or a fragment thereof having lysinactivity, wherein the encoded lysin polypeptide has the property ofinhibiting the growth, or reducing the population, or killing P.aeruginosa, and optionally at least one other species of Gram-negativebacteria, the nucleic acid being operatively linked to a heterologouspromoter.

In some embodiments, the nucleic acid sequence is a cDNA sequence.

In yet another aspect, the disclosure provides an isolatedpolynucleotide comprising a nucleic acid molecule that encodes a lysinpolypeptide comprising an amino acid sequence having at least 80% or atleast 85% or at least 90% or at least 95% sequence identity to asequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO:10, or a fragment thereof having lysin activity, wherein the encodedlysin polypeptide inhibits the growth, or reduces the population, orkills P. aeruginosa and optionally at least one other species ofGram-negative bacteria.

In some embodiments, the polynucleotide is cDNA.

In another aspect, the disclosure provides a method of inhibiting thegrowth, or reducing the population, or killing of at least one speciesof Gram-negative bacteria, the method comprising contacting the bacteriawith a composition containing an effective amount of a lysin polypeptidecomprising an amino acid sequence at least 80% identical to polypeptidesequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO:10, or active fragments thereof, wherein the lysin polypeptide has theproperty of inhibiting the growth, or reducing the population, orkilling P. aeruginosa and optionally at least one other species ofGram-negative bacteria.

In a related aspect, the disclosure provides a method of treating abacterial infection caused by a Gram-negative bacteria selected from thegroup consisting of P. aeruginosa and optionally one or more additionalspecies of Gram-negative bacteria, comprising administering to a subjectdiagnosed with, at risk for, or exhibiting symptoms of a bacterialinfection, a composition containing an effective amount of a lysinpolypeptide comprising an amino acid sequence at least 80% identical toa polypeptide sequence selected from the group consisting of SEQ ID NO:1-SEQ ID NO: 10, or an active fragment thereof, wherein the lysinpolypeptide has the property of inhibiting the growth, or reducing thepopulation, or killing P. aeruginosa and optionally at least one otherspecies of Gram-negative bacteria.

In another aspect, the disclosure provides a method of treating atopical or systemic pathogenic bacterial infection caused by aGram-negative bacteria selected from the group consisting of P.aeruginosa and optionally one or more additional species ofGram-negative bacteria in a subject, comprising administering to asubject composition containing an effective amount of a lysinpolypeptide comprising an amino acid sequence at least 80% identical topolypeptide sequence selected from the group consisting of SEQ ID NO:1-SEQ ID NO: 15, wherein the polypeptide or peptide has the property ofinhibiting the growth, or reducing the population, or killing P.aeruginosa and optionally at least one other Gram-negative bacteria.

In yet another aspect, the disclosure provides a method of preventing ortreating a bacterial infection comprising co-administering to a subjectdiagnosed with, at risk for, or exhibiting symptoms of a bacterialinfection, a combination of a first effective amount of the compositioncontaining an effective amount of lysin polypeptide comprising aminoacid sequence at least 80%, or at least 85%, or at least 90%, or atleast 95% identical to polypeptide sequence selected from the groupconsisting of SEQ ID NO: 1-SEQ ID NO: 10, or fragments thereof, and asecond effective amount of an antibiotic suitable for the treatment ofGram-negative bacterial infection.

In some embodiments, Gram-negative bacteria is selected from the groupconsisting of Pseudomanas aeruginosa, Klebsiella spp., Enterobacterspp., Escherichia coli, Citrobacter freundii, Salmonella typhimurium,Yersinia pestis, and Franciscella tulerensis.

In some embodiments, lysin polypeptide amino acid sequence is at least85% or at least 90% identical to a sequence selected from the groupconsisting of SEQ ID NO: 1-SEQ ID NO: 10, or a fragment thereof havinglysin activity.

In some embodiments, the lysin polypeptide amino acid sequence is atleast 95% identical to a sequence selected from the group consisting ofSEQ ID NO: 1-SEQ ID NO: 10, or a fragment thereof having lysin activity.

In some embodiments, the Gram-negative bacterial infection is aninfection caused by Pseudomonas aeruginosa.

In some embodiments, the antibiotic is selected from one or more ofceftazidime, cefepime, cefoperazone, ceftobiprole, ciprofloxacin,levofloxacin, aminoglycosides, imipenem; meropenem, doripenem,gentamicin; tobramycin; amikacin; piperacillin, ticarcillin, penicillin,rifampicin, polymyxin B, and colistin.

In another aspect, the disclosure provides a method for augmenting theefficacy of an antibiotic suitable for the treatment of Gram-negativebacterial infection, comprising co-administering the antibiotic incombination with one or more lysin polypeptides comprising an amino acidsequence at least 80% identical to a polypeptide sequence selected fromthe group consisting of SEQ ID NO: 1-SEQ ID NO: 10, or an activefragment thereof, wherein administration of the combination is moreeffective in inhibiting the growth, or reducing the population, orkilling the Gram-negative bacteria than administration of either theantibiotic or the lysin polypeptide or active fragment thereofindividually.

In some embodiments, the lysin polypeptide amino acid sequence is atleast 90% identical to a sequence selected from the group consisting ofSEQ ID NO: 1-SEQ ID NO: 10, or a fragment thereof having lysin activity.

In some embodiments, the lysin polypeptide amino acid sequence is atleast 95% identical to a sequence selected from the group consisting ofSEQ ID NO: 1-SEQ ID NO: 10, or a fragment thereof having lysin activity.

In another aspect, an isolated lysin polypeptide, comprising an aminoacid sequence having at least 80% or at least 85% or at least 90% or atleast 95% identity to a polypeptide sequence selected from the groupconsisting of SEQ ID NO: 1-SEQ ID NO: 10, or a fragment thereof havinglysin activity, wherein the lysin polypeptide inhibits the growth, orreduces the population, or kills P. aeruginosa and, optionally, at leastone other species of Gram-negative bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the nucleotide and amino acid sequences of lysinpolypeptide GN37 reported in the present disclosure. FIG. 1A (left) isan amino acid sequence of GN37. FIG. 1A (right) is a nucleotide sequenceof GN37. FIG. 1B is a schematic of GN37, indicating that GN37 is amember of peptidase M15_4 family of PGHs with DD- andDL-carboxypeptidase activities (including members of the VanYsuperfamily). FIG. 1C shows a multiple sequence alignment comparing GN37to a Gram-positive partial homolog (Streptomyces, GenBank sequenceAGJ50592.1) and to putative or confirmed endolysins from otherGram-negative pathogens including E. coli (GenBank WP_001117823.1 andNP_543082.1—both putative endolysins), Yersinia spp. (GenBankCAJ28446.1-confirmed endolysin) and Acinetobacter baumannii, (GenBankWP_034684053.1, putative lysin).

FIG. 2 provides amino acid (bold font) and nucleotide (regular font)sequences of lysin polypeptides GN2, GN4, GN14, and GN43 reported in thepresent disclosure.

FIG. 3 is a bar graph depicting fold induction of fluorescence signalover control by P. aeruginosa strain PAO1 in the presence of various GNlysin polypeptides, wherein fluorescence indicates outer membranepermeabilization.

FIG. 4 is a bar graph showing antibacterial activity of GN lysinpolypeptides against P. aeruginosa strain PAO1. The reduction in colonyforming units (CFU) is presented on a logarithmic scale.

FIG. 5 provides amino acid sequences of five lysin peptides derived fromGN4: PGN4, FGN4-1, FGN4-2, FGN4-3, and FGN4-4.

FIG. 6 is a bar graph showing the antibacterial activity of eachGN4-derived lysin peptide (PGN4, FGN4-1, FGN4-2, FGN4-3, and FGN4-4)against P. aeruginosa strain PAO1. The reduction in CFU counts ispresented along a logarithmic scale.

FIG. 7 is a bar graph showing the antibacterial activity in human serumof pooled GN lysin polypeptides and GN4-derived lysin peptides of thepresent disclosure against P. aeruginosa strain PAO1. The reduction inCFU counts is presented along a logarithmic scale.

DETAILED DESCRIPTION

Definitions

As used herein, the following terms and cognates thereof shall have themeanings ascribed to them below unless the context clearly indicatesotherwise.

“Gram-negative bacteria” generally refers to bacteria which produce acrystal violet stain that is decolorized in Gram staining, i. e. they dohot retain crystal violet dye in the Gram staining protocol. As usedherein, the term “Gram-negative bacteria” may describe withoutlimitation one or more (i.e., one or a combination) of the followingbacterial species: Acinetobacter baumannii, Acinetobacter haemolyticus,Actinobacillus actinomycetemcomitans, Aeromonas hydrophila, Bacteroidesfragilis, Bacteroides theataioatamicron, Bacteroides distasonis,Bacteroides ovatus, Bacteroides vulgatus, Bordetella pertussis, Brucellamelitensis, Burkholderia cepacia, Burkholderia pseudomallei,Burkholderia mallei, Prevotella corporis, Prevotella intermedia,Prevotella endodontalis, Porphyromonas asacchitrolytica, Campylobacterjejuni, Campylobacter coli, Campylobacter fetus, Citrobacter freundii,Citrobacter koseri, Edwarsiella tarda, Eikenella corrodens, Enterobactercloacae, Enterobacter aerogeries, Enterobacter agglomerans, Escherichiacoli, Francisella tularensis, Haemophilus influenzae, Haemophilusducreyi, Helicobacter pylori, Kingella kingae, Klebsiella pneumoniae,Klebsiella oxytoca, Klebsiella rhinoscleromatis, Klebsiella ozaenae,Legionella pemimophila, Moraxella catarrhalis, Morganella morganii,Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida,Plesiomonas shigelloides, Proteus mirabilis, Proteus vulgaris, Proteuspenneri, Proteus myxofaciens, Providencia stuartii, Providenciarettgeri, Providencia alcalifaciens, Pseudomonas aeruginosa, Pseudomonasfluorescens, Salmonella typhi, Salmonella paratyphi, Serratiamarcescens, Shigella flexneri, Shigella boydii, Shigella sonnei,Shigella dysenteriae, Stenotrophomonas maltophilia, Streptobacillusmoniliformis, Vibrio cholerae, Vibrio parahaemolyticus, Vibriovulnificus, Vibrio alginolyticus, Yersinia enterocolitica, Yersiniapestis, Yersinia pseudotuberculosis, Chlamydophila pneumoniae,Chlamydophila trachomatis, Ricketsia prowazekii, Coxiella burnetii,Ehrlichia chaffeensis, or Bartonella hensenae. The compounds of thepresent disclosure will be useful in preventing or inhibiting pathogenicbacterial growth and in treating one or more bacterial infections,particularly but not necessarily exclusively involving Gram-negativebacteria and notably P. aeruginosa.

The term “bactericidal” in the context of an agent conventionally meanshaving the property of causing the death of bacteria or capable ofkilling bacteria to an extent of at least a 3-log (99.9%) or betterreduction among an initial population of bacteria.

The term “bacteriostatic” conventionally means having the property ofinhibiting bacterial growth, including inhibiting growing bacterialcells, thus causing a 2-log (99%) or better and up to just under a 3-logreduction among an initial population of bacteria.

The term “antibacterial” in a context of an agent is used generically toinclude both bacteriostatic and bactericidal agents.

The term “drug resistant” in a context of a pathogen and morespecifically a bacterium, generally refers to a bacterium that isresistant to the antimicrobial activity of a drug. When used in a moreparticular way, drug resistance specifically refers to antibioticresistance. In some cases, a bacterium that is generally susceptible toa particular antibiotic can develop resistance to the antibiotic,thereby becoming a drug resistant microbe or strain. A “multi-drugresistant” pathogen is one that has developed resistance to at least twoclasses of antimicrobial drugs, each used as monotherapy. For example,certain strains of Pseudomonas aeruginosa have been found to beresistant to nearly all or all antibiotics including aminoglycosides,cephalosporins, fluoroquinolones, and carbapenems (Antibiotic ResistantThreats in the United States, 2013, U.S. Department of Health andServices, Centers for Disease Control and Prevention). One skilled inthe art can readily determine if a bacterium is drug resistant usingroutine laboratory techniques that determine the susceptibility orresistance of a bacterium to a drug or antibiotic.

The term “pharmaceutically acceptable carrier” includes any and allsolvents, additives, excipients, dispersion media, solubilizing agents,coatings, preservatives, isotonic and absorption delaying agents,surfactants, propellants and the like that are physiologicallycompatible. The carrier(s) must be “acceptable” in the sense of notbeing deleterious to the subject to be treated in amounts typically usedin medicaments. Pharmaceutically acceptable carriers are compatible withthe other ingredients of the composition without rendering thecomposition unsuitable for its intended purpose. Furthermore,pharmaceutically acceptable carriers are suitable for use with subjectsas provided Herein without undue adverse side effects (such as toxicity,irritation, and allergic response). Side effects are “undue” when theirrisk outweighs the benefit provided by the composition. Non-limitingexamples of pharmaceutically acceptable carriers or excipients includeany of the standard pharmaceutical carriers such as phosphate bufferedsaline solutions, water, and emulsions such as oil/water emulsions andmicroemulsions.

For solid compositions comprising a lyophilized lysin polypeptide,excipients such as urea or mesna can be included to improve stability.Other excipients include bulking agents, buffering agents, tonicitymodifiers, surfactants, preservatives and co-solvents.

For solid oral compositions comprising lysin polypeptide, suitablepharmaceutically acceptable excipients include, but are not limited to,starches, sugars, diluents, granulating agents, lubricants, binders,disintegrating agents and the like.

For liquid oral compositions, suitable pharmaceutically acceptableexcipients include, but are not limited to, water, glycols, oils,alcohols, flavoring agents, preservatives, and the like.

For topical solid compositions such as creams, gels, foams, ointments,or sprays, suitable excipients include, but are not limited to a cream,a cellulosic or oily base, emulsifying agents, stiffening agents,rheology modifiers or thickeners, surfactants, emollients,preservatives, humectants, alkalizing or buffering agents, and solvents.

Suitable excipients for the formulation of the foam base include, butare not limited to, propylene glycol, emulsifying wax, cetyl alcohol,and glyceryl stearate. Potential preservatives include methylparaben andpropylparaben.

The term “effective amount” refers to an amount which, when applied oradministered in an appropriate frequency or dosing regimen, issufficient to prevent or inhibit bacterial growth or prevent, reduce orameliorate the onset, severity, duration or progression of the disorderbeing treated (here bacterial pathogen growth or infection), prevent theadvancement of the disorder being treated, cause the regression of thedisorder being treated, or enhance or improve the prophylactic ortherapeutic effect(s) of another therapy, such as antibiotic orbacteriostatic therapy.

The term “co-administer” is intended to embrace separate administrationof a lysin polypeptide and an antibiotic or any other antibacterialagent in a sequential manner as well as administration of these agentsin a substantially simultaneous manner, such as in a singlemixture/composition or in doses given separately, but nonethelessadministered substantially simultaneously to the subject, for example atdifferent times in the same day or 24-hour period. Suchco-administration of lysin polypeptides with one or more additionalantibacterial agents can be provided as a continuous treatment lastingup to days, weeks, or months. Additionally, depending on the use, theco-administration need not be continuous or co-extensive. For example,if the use were as a topical antibacterial agent to treat, e.g., abacterial ulcer or an infected diabetic ulcer, the lysin could beadministered only initially within 24 hours of the first antibiotic useand then the antibiotic use may continue without further administrationof lysin.

The term “subject” refers to a subject to be treated and includes interalia a mammal, a plant, a lower animal, a single cell organism or a cellculture. For example, the term “subject” is intended to includeorganisms, e.g., prokaryotes and eukaryotes, which are susceptibe to orafflicted with Gram-negative bacterial infections. Examples of subjectsinclude mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats,cats, mice, rabbits, rats, and transgenic non-human animals. In certainembodiments, the subject is a human, e.g., a Human suffering from, atrisk of suffering from, or susceptible to a Gram-negative bacterialinfection, whether such infection be systemic or confined to aparticular organ or tissue.

The term “polypeptide” is used interchangeably with the term “protein”and “peptide” and refers to a polymer made from amino acid residues andhaving at least about 30 amino acid residues. The term includes not onlypolypeptides in isolated form, but also active fragments and derivativesthereof (defined below). The term “polypeptide” also encompasses fusionproteins or fusion polypeptides comprising a lysin polypeptide asdescribed below and maintaining the lysin function. A polypeptide can bea naturally occurring polypeptide or an engineered or syntheticallyproduced polypeptide. A particular lysin polypeptide can be, forexample, derived or removed from a native protein by enzymatic orchemical cleavage, or can be prepared using conventional peptidesynthesis techniques (e.g., solid phase synthesis) or molecular biologytechniques (such as those disclosed in Sambrook, J. et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989)) or can be strategically truncated or segmentedyielding active fragments, as illustrated for example herein with afragment of GN4 comprising the amphipathic domain of GN4 and furthertruncated versions thereof maintaining lysin activity against the sameof at least one common target bacterium. Variants of native lysinpolypeptides are also encompassed having at least 80% or at least 85% orat least 90% or at least 95% or at least 98% sequence identity with thenative lysin polypeptide (which, as stated above includes activefragments of a native lysin protein).

The term “fusion polypeptide” refers to an expression product resultingfrom the fusion of two or more nucleic acid segments, resulting in afused expression product typically having two domains or segments withdifferent properties or functionality. In a more particular sense, theterm “fusion polypeptide” also refers to a polypeptide or peptidecomprising two or more heterologous polypeptides or peptides covalentlylinked, either directly or via an amino acid or peptide linker. Thepolypeptides forming the fusion polypeptide are typically linkedC-terminus to N-terminus, although they can also be linked C-terminus toC-terminus, N-terminus to N-terminus, of N-terminus to C-terminus. Theterm “fusion polypeptide” can be used interchangeably with the term“fusion protein.” Thus the open-ended expression “a polypeptidecomprising” a certain structure includes larger molecules than therecited structure such as fusion polypeptides.

The term “heterologous” refers to nucleotide, peptide, or polypeptidesequences that are not naturally contiguous. For example, in the contextof the present disclosure, the term “heterologous” can be used todescribe a combination or fusion of two or more peptides and/orpolypeptides wherein the fusion peptide or polypeptide is not normallyfound in nature, such as for example a lysin polypeptide or activefragment thereof and a cationic and/or a polycationic peptide, anamphipathic peptide, a sushi peptide (Ding et al. Cell Mol Life Sci.,65(7-8):1202-19 (2008)), a defensin peptide (Ganz, T. Nature ReviewsImmunology 3, 710-720 (2003)), a hydrophobic peptide and/or anantimicrobial peptide which may have enhanced lysin activity. Includedin this definition are two or more lysin polypeptides or activefragments thereof. These can be used to make a fusion polypeptide withlysin activity

The term “active fragment” refers to a portion of a full-lengthpolypeptide disclosed herein which retains one or more functions orbiological activities of the isolated original polypeptide. See forexample GN4 in FIG. 2 and fragments thereof in FIG. 5 (FGN4-1 andFGN4-2). A biological activity of particular interest herein is that ofa lysin active to bore through the outer membrane and hydrolyze thecoating of Gram-negative bacteria, whether by cleaving a sugar backboneor peptide bond.

The term “amphipathic peptide” refers to a peptide having bothhydrophilic and hydrophobic functional groups. Preferably, secondarystructure places hydrophobic and hydrophilic amino acid residues atdifferent ends of the peptide. These peptides often adopt a helicalsecondary structure.

The term “cationic peptide” refers to a peptide having positivelycharged amino acid residues. Preferably, a cationic peptide has apKa-value of 9.0 or greater. The term “cationic peptide” in the contextof the present disclosure also encompasses polycationic peptides.

The term “polycationic peptide” as used herein refers to a syntheticallyproduced peptide composed of mostly positively charged amino acidresidues, in particular lysine and/or arginine residues. The amino acidresidues that are not positively charged can be neutrally charged aminoacid residues and/or negatively charged amino acid residues and/orhydrophobic amino acid residues.

The term “hydrophobic group” refers to a chemical group such as ah aminoacid side chain which has low or no affinity for water molecules buthigher affinity for oil molecules. Hydrophobic substances tend to havelow or no solubility in water or aqueous phases and are typically apolarbut tend to have higher solubility in oil phases. Examples ofhydrophobic amino acids include glycine (Gly), alanine (Ala), valine(Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine(Phe), methionine (Met), and tryptophan (Trp).

The term “augmenting” within the context of the present disclosure meansthat a degree of antimicrobial activity is higher than it would beotherwise. “Augmenting” encompasses additive as well as synergistic(superadditive) effects.

The term “synergistic” or “superadditive” in relation to an effect meansa beneficial effect brought about by two active substances that exceedsthat produced by each substance administered or applied alone. One orboth active ingredients may be employed at a subtreshold level, i.e., alevel at which if the active substance is employed individually producesno or a very limited effect.

The term “treatment” refers to any process, action, application,therapy, or the like, wherein a subject, including a human beings issubjected to medical aid with the object of curing a disorder, oreradicating a pathogen, or improving the subject's condition, directlyor indirectly. Treatment also refers to reducing incidence, oralleviating symptoms, eliminating recurrence, preventing recurrence,preventing incidence, improving symptoms, improving prognosis orcombinations thereof. “Treatment” further encompasses reducing thepopulation, growth rate or virulence of the bacteria in the subject andthereby controlling or reducing a bacterial infection in a subject orbacterial contamination of an organ or tissue or environment. Thus“treatment” that reduces incidence is effective to inhibit growth of atleast one Gram-negative bacterium in a particular milieu, whether it bea subject or an environment. On the other hand “treatment” of an alreadyestablished infection refers to reducing the population or killing,including even eradicating the Gram-negative bacteria responsible for aninfection or contamination.

The term “preventing” includes the prevention of the incidence,recurrence, spread, onset or establishment of a disorder such as abacterial infection. It is not intended that the present disclosure belimited to complete prevention or to prevention of establishment of aninfection. In some embodiments, the onset is delayed, or the severity ofa subsequently contracted disease is reduced, and such constituteexamples of prevention. Contracted diseases in the context of thepresent disclosure encompass both those manifesting with clinical orsubclinical symptoms, such as the detection of as well as the detectionof growth of a bacterial pathogen when symptoms associated with suchpathology are not yet manifest.

The term “derivative” in the context of a peptide or polypeptide (whichas stated herein includes an active fragment) is intended to encompassfor example, a polypeptide modified to contain one or more-chemicalmoieties other than an amino acid that do not substantially adverselyimpact or destroy the lysin activity. The chemical moiety can be linkedcovalently to the peptide, e.g., via an amino terminal amino acidresidue, a carboxy terminal amino acid residue, of at an internal aminoacid residue. Such modifications include the addition of a protective orcapping group on a reactive moiety, addition of a detectable label, suchas antibody and/or fluorescent label, addition or modification ofglycosylation, or addition of a bulking group such as PEG (pegylation)and other changes that do not substantially adversely impact or destroythe activity of the lysin polypeptide.

Commonly used protective groups that may be added to lysin polypeptidesinclude, but are not limited to t-Boc and Fmoc.

Commonly used fluorescent label proteins such as, but not limited to,green fluorescent protein (GFP), red fluorescent protein (RFP), cyanfluorescent protein (CFP), yellow fluorescent protein (YFP) and mCherry,are compact proteins that can be bound covalently or noncovalently to alysin polypeptide or fused to a lysin polypeptide without interferingwith normal functions of cellular proteins. Typically, a polynucleotideencoding a fluorescent protein is inserted upstream or downstream of thelysin polynucleotide sequence. This will produce a fusion protein (e.g.,Lysin Polypeptide GFP) that does not interfere with cellular function orfunction of a lysin polypeptide to which it is attached.

Polyethylene glycol (PEG) conjugation to proteins has been used as amethod for extending the circulating half-life of many pharmaceuticalproteins. Thus, in the context of lysin polypeptide derivatives, theterm “derivative” encompasses lysin polypeptides chemically modified bycovalent attachment of one or more PEG molecules. It is anticipated thatpegylated lysin polypeptides will exhibit prolonged circulationhalf-life compared to the unpegylated lysin polypeptides, whileretaining biological and therapeutic activity.

The term “percent amino acid sequence identity” with respect to thelysin polypeptide sequences is defined herein as the percentage of aminoacid residues in a candidate sequence that are identical with the aminoacid residues in the specific lysin polypeptide sequence, after aligningthe sequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for example, usingpublicly available software such as BLAST or Megalign (DNASTAR)software. Two or more polypeptide sequences can be anywhere from 0-100%identical, or any integer value there between. In the context of thepresent disclosure, two polypeptides are “substantially identical” whenat least 80% of the amino acid residues (preferably at least about 85%,at least about 90%, and preferably at least about 95%) are identical.The term “percent (%) amino acid sequence identity” as described hereinapplies to lysin peptides as well. Thus, the term “substantiallyidentical” will encompass mutated, truncated, fused, or otherwisesequence-modified variants of isolated lysin polypeptides and peptidesdescribed herein, and active fragments thereof, as well as polypeptideswith substantial sequence identity (e.g., at least 80%, at least 85%, atleast 90%, or at least 95% identity as measured for example by one ormore methods referenced above) as compared to the reference polypeptide.

Two amino acid sequences are “substantially homologous” when at leastabout 80% of the amino acid residues (preferably at least about 85%, atleast about 90%, and preferably at least about 95%) are identical, orrepresent conservative substitutions. The sequences of lysinpolypeptides of the present disclosure, are substantially homologouswhen one or more, or several, or up to 10%, or up to 15%, or up to 20%of the amino acids of the lysin polypeptide are substituted with asimilar or conservative amino acid substitution, and wherein theresulting lysin have the profile of activities, antibacterial effects,and/or bacterial specificities of lysin polypeptides disclosed herein.The meaning of “substantially homologous” described herein applies tolysin peptides as well.

The term “inhalable composition” refers to pharmaceutical compositionsof the present disclosure that are formulated for direct delivery to therespiratory tract during or in conjunction with routine or assistedrespiration (e.g., by intra tracheobronchial, pulmonary, and/or nasaladministration), including, but not limited to, atomized, nebulized, drypowder and/or aerosolized formulations.

The term “biofilm” refers to bacteria that attach to surfaces andaggregate in a hydrated polymeric matrix of their own synthesis. Abiofilm is an aggregate of microorganisms in which cells adhere to eachother on a surface. These adherent cells are frequently embedded withina self-produced matrix of extracellular polymeric substance (EPS).Biofilm EPS, which is also referred to as slime (although not everythingdescribed as slime is a biofilm) or plaque, is a polymericconglomeration generally composed of extracellular DNA, proteins, andpolysaccharides.

The term “suitable” in the context of an antibiotic being suitable foruse against certain bacteria refers to an antibiotic that was found tobe effective against those bacteria even if resistance subsequentlydeveloped.

The term “antimicrobial peptide” (AMR) refers to a member of a widerange of short (generally 6 to 50 amino acid residues in length)cationic, gene encoded peptide antibiotics that can be found invirtually every organism. Different AMPs display different properties,and many peptides in this class are being intensively researched hotonly as antibiotics, but also as templates for cell penetratingpeptides. Despite sharing a few common features (e.g., cationicity,amphipathicity and short size), AMP sequences vary greatly, and at leastfour structural groups (alpha.-helical, beta.-sheet, extended andlooped) have been proposed to accommodate the diversity of the observedAMP conformations. Likewise, several modes of action as antibiotics havebeen proposed, and it was shown e.g. that the primary target of many ofthese peptides is the cell membrane whereas for other peptides theprimary target is cytoplasmic invasion and disruption of core metabolicfunctions. AMPs may become concentrated enough to exhibit cooperativeactivity despite the absence of specific target binding; for example, byforming a pore in the membrane, as is the case for most AMPs. However,this phenomenon has only been observed in model phospholipid bilayers,and in some cases, AMP concentrations in the membrane that were as highas one peptide molecule per six phospholipid molecules were required forthese events to occur. These concentrations are close to, if not at,full membrane saturation. As the minimum, inhibitory concentration (MIC)for AMPs are typically in the low micromolar range, scepticism hasunderstandably arisen regarding the relevance of these thresholds andtheir importance in vivo (Melo et al., Nature Reviews Microbiology, 7,245-250 (2009)).

Defensins are a large family of small, cationic, cysteine- andarginine-rich antimicrobial peptides, found in both vertebrates andinvertebrates (Wilmes, M. and Sahl, H., Int J Med Microbiol.;304(1):93-9 (2014)). Defensins are divided into five groups according tothe spacing pattern of cysteines: plant, invertebrate, alpha-, beta-,and theta-defensins. The latter three are mostly found in mammals,alpha-defensins are proteins found in neutrophils and intestinalepithelia. Beta-defensins are the most widely distributed and aresecreted by leukocytes and epithelial cells of many kindstheta-defensins have been rarely found so far e.g. in leukocytes ofrhesus macaques. Defensins are active against bacteria, fungi and manyenveloped and nonenveloped viruses. However, the concentrations neededfor efficient killing of bacteria are mostly high, i.e. in themicromolar range. Activity of many peptides may be limited in thepresence of physiological salt conditions, divalent cations and serum.In addition, defensins often have hemolytic activity which is notdesirable for the products and methods of the present disclosure.

Sushi peptides are characterized by the presence of sushi domains, alsoknown as complement control protein (CCP) modules or short consensusrepeats (SCR)). Sushi domains are found in a variety of complement andadhesion proteins, which contain tandem arrangements of Sushi domainsinterspersed by short linking sequences. Sushi domains contain aconsensus sequence spanning approximately 60 residues, which in turncontains four invariant cysteine residues that are involved inintramolecular disulphide bonds, a highly conserved tryptophan, andconserved glycine, proline and hydrophobic residues (Kirkitadze, M. andBarlow, P., Immunol Rev, 180:146-61 (2001)). Sushi domains are known tobe involved in protein-protein and protein-ligand interactions. Peptidescontaining a Sushi domain have been shown to have antimicrobialactivities (Ding, J L. and Ho, B. Drug Development Research, 62:317-335(2004)).

Cathelicidins are multifunctional antimicrobial peptides also known ascationic host-defence peptides (CHDP)—a class of peptides proposed asantimicrobial therapeutics—and an important component of innate hostdefence against infection. In addition to microbicidal potential, thesepeptides have properties with the capacity to modulate inflammation andimmunity. Recently, the delivery of exogenous human cathelicidin LL-37was found to enhance a protective pro-inflammatory response to infectionin a murine model of acute P. aeruginosa lung infection, demonstratingcathelicidin-mediated enhancement of bacterial clearance in vivo(Beaumont et al. PLoS One. 2;9(6):e99029 (2014)). Thus, cathelidicineffectively promoted bacterial clearance from the lung in the absence ofdirect microbicidal activity, with an enhanced early neutrophil responsethat required both infection and peptide exposure and was independent ofnative cathelicidin production. Furthermore, althoughcathelicidin-deficient mice had an intact early cellular inflammatoryresponse, later phase neutrophil response to infection was absent inthese animals, with significantly impaired clearance of P. aeruginosa.These findings demonstrated the importance of the modulatory propertiesof cathelicidins in pulmonary infection in vivo and highlighted a keyrole for cathelicidins in the induction of protective pulmonaryneutrophil responses, specific to the infectious milieu. Beaumont, P. E.et al, PLoS One. 2014; 9(6); e99029. Published online 2014 Jun. 2, doi:10.1371/journal.pone.0099029.

EMBODIMENTS

The present disclosure relates to new antibacterial agents againstGram-negative bacteria. In particular, the present disclosure relates tolysin polypeptides (including active fragments thereof) active againstGram-negative bacteria, such as Pseudomonas aeruginosa. Examples of suchlysin polypeptides are those having an amino acid sequence within theset SEQ ID NO: 1-SEQ ID NO: 10. The native sequences were identified bybioinformatics techniques from a previously sequenced but partiallyelucidated phage genome. Although some of the sequences thus identifiedhad been annotated as putative endolysins, no function had beenpreviously definitively attributed to polypeptides having thesesequences. Moreover, several sequences annotated as putativeendolysisns, on synthesis or expression, turned out to be wholly devoidof lysin activity or inactive against the target pathogen. On isolation,expression and testing, only a handful of the bioinformaticallyidentified sequences in fact had Gram-negative lysin function.Additionally, active fragments of the lysins were identified andsequence-modified active peptides and polypeptides having Gram-negativelysin activity were prepared. Furthermore, in accordance with thepresent disclosure, such sequence modified papetides include fragmentsof the confirmed native Gram-negative lysin polypeptides maintaininglysin activity, as well as variants thereof having 80% or more (such asat least 85%, at least 90% at least 85% or at least 98%) sequenceidentity with the native lysin polypeptides or active fragments thereofand indeed the nonidentical portions might include substitutions withboth natural and non-natural (synthetic) amino acid residues. Thepresent inventors have determined that the alpha helical domain of theC-terminal end of these polypeptides is important for Gram-negativelysin activity and have conducted studies to pinpoint the activity, butany peptide with a sequence that is 80% or more (such as 85%, 90%, 95%or 98% or 99%) identical to the native lysins disclosed herein orfragments thereof can be quickly tested for activity againstGram-negative bacteria including P. aeruginosa, and others such asKlebsiella spp., Enterobacter spp., Escherichia coli, Citrobacterfreundii, Salmonella typhimurium, Yersinia pestis, and Franciscellatulerensis. Such testing can follow for example the teachings providedin Examples 2, 3, 4, or Prophetic Example 1. Of course, the testingprocedures and protocols themselves are not limited to those in theseExamples but can be any methods known to those skilled in the art forassessing effectiveness of an antibacterial and indeed an antimicronbialagent.

In one embodiment, the present disclosure provides methods for treatmentof a bacterial infection in a subject caused by Gram-negative bacteriacomprising administering to the subject an effective amount of a lysinpolypeptide having at least 80% or at least 85% or at least 90% or atleast 95% amino acid sequence identity to SEQ ID NO: 1 through SEQ IDNO: 10. The bacteria may be selected from the group consisting ofAcinetobacter baumannii, Acinetobacter haemolyticus, Actinobacillusactinomycetemcomitans, Aeromonas hydrophila, Bacteroides fragilis,Bacteroides theataioatamicron, Bacteroides distasonis, Bacteroidesovatus, Bacteroides vulgatus, Bordetella pertussis, Brucella melitensis,Burkholderia cepacia, Burkholderia pseudomallei, Burkholderia mallei,Fusobacterium, Prevotella corporis, Prevotella intermedia, Prevotellaendodontalis, Porphyromonas asaccharolytica, Campylobacter jejuni,Campylobacter fetus, Campylobacter coli, Citrobacter freundii,Citrobacter koseri, Edwarsiella tarda, Eikenella corrodens, Enterobactercloacae, Enterobacter aerogenes, Enterobacter agglomerans, Escherichiacoli, Francisella tularensis, Haemophilus influenzae, Haemophilusducreyi, Helicobacter pylori, Kingella kingae, Klebsiella pneumoniae,Klebsiella oxytoca, Klebsiella rhinoscleromatis, Klebsiella ozaenae,Legionella penumophila, Moraxella catarrhalis, Morganella morganii,Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida,Plesiomonas shigelloides, Proteus mirabilis, Proteus vulgaris, Proteuspenneri, Proteus myxofaciens, Providencia stuartii, Providenciarettgeri, Providencia alcalifaciens, Pseudomonas aeruginosa, Pseudomonasfluorescens, Salmonella typhi, Salmonella paratyphi, Serratiamarcescens, Shigella flexneri, Shigella boydii, Shigella sonnei,Shigella dysenteriae, Stenotrophomonas maltophilia, Streptobacillusmoniliformis, Vibrio cholerae, Vibrio parahaemolyticus, Vibriovulnificus, Vibrio alginolyticus, Yersinia enterocolitica, Yersiniapestis, Yersinia pseudotuberculosis, Chlamydia pneumoniae, Chlamydiatrachomatis, Ricketsia prowazekii, Coxiella burnetii, Ehrlichiachafeensis, and Bartonella hensenae. For example, in a particularembodiment, the Gram-negative bacterial infection is an infection causedbacteria selected from the group consisting of Acinetobacter baumannii,Bordetella pertussis, Burkholderia cepacia, Burkholderia pseudomallei,Burkholderia mallei, Campylobacter jejuni, Campylobacter coli,Enterobacter cloacae, Enterobacter aerogenes, Escherichia coli,Francisella tularensis, Haemophilus influenzae, Haemophilus ducreyi,Helicobacter pylori, Klebsiella pneumoniae, Legionella penumophila,Moraxella catarrhalis, Morganella morganii, Neisseria gonorrhoeae,Neisseria meningitidis, Pasteurella multocida, Proteus mirabilis,Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhi, Serratiamarcescens, Shigella flexneri, Shigella boydii, Shigella sonnei,Shigella dysenteriae, Stenotrophomonas maltophilia, Vibrio cholerae, andChlamydia pneumoniae. In a specific embodiment, the Gram-negativebacterial infection is an infection caused by one or more of bacteriaselected from the group consisting of Salmonella typhimurium, Salmonellatyphi, Shigella spp., Escherichia coli, Acinetobacter baumanii,Pseudomonas aeruginosa, Klebsiella pneumonia, Neisseria gonorrhoeae,Neisseria meningitides, Serratia spp. Proteus mirabilis, Morganellamorganii, Providencia spp., Edwardsiella spp., Yersinia spp.,Haemophilus influenza, Bartonella quintana, Brucella spp., Bordetellapertussis, Burkholderia spp., Moraxella spp., Francisella tularensis,Legionella pneumophila, Coxiella burnetii, Bacteroides spp.,Enterobacter spp., and Chlamydia spp.

Based on (i) the fact that the lysin peptides and polypeptides of thepresent disclosure are able to bore through the OM of Gram-negativebacteria and reach their substrate, killing such bacteria andsubstantially reducing the rate of growth of bacterial colonies, and(ii) on similar observations with other lysin polypeptides that areengineered to penetrate the OM in buffer and media such as artilysins,it is anticipated that the lysin polypeptides of the present disclosurewill be useful in treating one or more Gram-negative bacterialinfections. Moreover, the fact that the present lysin polypeptidespossess activity against Gram-negative targets even before fusion (ifany) with cationic and other antimicrobial peptides) may be advantageousover artilysisns as the latter appear to be inhibited by human sera.Deslouches, B. et al, Activity of the De Novo Engineered AntimicrobialPeptide WLBU2 against Pseudomonas aeruginosa in Human Serum and WholeBlood: Implications for Systemic Applications, Antimicrobial Agents &Chemotherapy, August 2005, p. 3208-3216 Vol. 49, No 8; Brogden N. et al,Int J Antimicrob Agents. 2011 September, 38(3): 217-225.doi:10.1016/j.ijantimicag.2011.05.004; Svenson, J. et al, J. Med. Chem.,2007, 50(14), pp 3334-3339.

In one embodiment, the terms “infection” and “bacterial infection” mayrefer inter alia to a respiratory tract infection (RTI), especially butnot exclusively to lower respiratory tract infections. In anotherembodiment, the terms “infection” and “bacterial infection” may refer toa sexually transmitted disease. In yet another embodiment, the terms“infection” and “bacterial infection” may refer to a urinary tractinfection. In a further embodiment, the terms “infection” and “bacterialinfection” may refer to acute exacerbation of chronic bronchitis (ACEB).In still another embodiment, the terms “infection” and “bacterialinfection” may refer to respiratory tract infections of patients havingcystic fibrosis (CF). In still a further embodiment, the terms“infection” and “bacterial infection” may refer to acute otitis media orneonatal septisemia. In yet a further embodiment, the terms “infection”and “bacterial infection” may refer to acute sinusitis. In oneembodiment, the terms “infection” and “bacterial infection” may refer toan infection caused by drug resistant bacteria even multidrug-resistantbacteria. In another embodiment, the terms “infection” and “bacterialinfection” may refer to catheter-related sepsis. In yet anotherembodiment, the terms “infection” and “bacterial infection” may refer tochlamydia. In a further embodiment, the terms “infection” and “bacterialinfection” may refer to community-acquired pneumonia (CAP) or tonosocomial respiratory tract infections. In still a further embodiment,the terms “infection” and “bacterial infection” may refer to acomplicated skin and skin structure infection. In yet a furtherembodiment, the terms “infection” and “bacterial infection” may refer touncomplicated skin and skin structure infections. In one embodiment, theterms “infection” and “bacterial infection” may refer to endocarditis.In another embodiment, the terms “infection” and “bacterial infection”may refer to febrile neutropenia. In still another embodiment, the terms“infection” and “bacterial infection” may refer to gonococcalcervicitis. In yet another embodiment, the terms “infection” and“bacterial infection” may refer to gonococcal urethritis. In a furtherembodiment, the terms “infection” and “bacterial infection” may refer tohospital-acquired pneumonia (HAP). In still a further embodiment, theterms “infection” and “bacterial infection” may refer to osteomyelitis.In yet a further embodiment, the terms “infection” and “bacterialinfection” may refer to sepsis. Common Gram-negative pathogens andassociated infections are listed in Table 1 of the present disclosure.These embodiments as well as pathogens and diseases listed in Table 1are meant to serve as examples of uses of the present methods and arenot intended to be limiting.

TABLE 1 Medically relevant Gram-negative bacteria and associateddiseases. Gram-negative pathogen Primary Disease/s Salmonellatyphimurium Gastrointestinal (GI) infections - salmoncllosis Shigellaspp. GI infections - shigellosis Escherichia coli Urinary tractinfections (UTIs) Acinetobacter baumanii Wound infections Pseudomonasaeruginosa bloodstream infections and pneumonia Klebsiella pneumoniaepneumonia, UTIs, and bloodstream infections Neisseria gonorrhoeaeSexually transmitted disease (STD) - gonorrhea Neisseria meningitidesMeningitis Serratia spp. Catheter contaminations, UTIs, and pneumoniaProteus mirabilis UTIs Morganella spp. UTIs Providencia spp. UTIsEdwardsiella spp. UTIs Salmonella typhi GI infections - typhoid feverYersinia pestis Bubonic and pneumonic plague Yersinia enterocolitica GIinfections Yersinia GI infections pseudotuberculosis Haemophilusinfluenza Meningitis Bartonella Quintana Trench fever Brucella spp.Brucellosis Bordetella pertussis Respiratory - Whooping coughBurkholderia spp. Respiratory Moraxella spp. Respiratory Francisellatularensis Tularemia Legionella pneumophila Respiratory - Legionnaires'disease Coxiella burnetii Q fever Bacteroides spp. Abdominal infectionsEnterobacter spp. UTIs and respiratory Chlamydia spp. STDs, respiratory,and ocular

In one embodiment, the present disclosure provides methods for treatmentof the Gram-negative bacterial infection in a subject caused byPseudomonas aeruginosa and optionally by at least one additional speciesof Gram-negative bacteria such as those selected from the groupconsisting of, Klebsiella spp., Enterobacter spp., Escherichia coli,Citrobacter freundii, Salmonella typhimurium, Yersinia pestis, andFranciscella tulerensis, which are the Gram-negative bacteria mostsignificant in human disease.

In one embodiment, the present disclosure provides methods for treatmentof the Gram-negative bacterial infection in a subject caused byPseudomonas aeruginosa. Pseudomonas aeruginosa (P. aeruginosa) is anoxidase-positive, Gram-negative, rod-shaped organism that is foundubiquitously in the environment. P. aeruginosa can grow in numeroushabitats, including but not limited to soil, water, and on plant andanimal tissue. It is an opportunistic organism and one of the mostproblematic nosocomial pathogens capable of causing localized orsystemic disease in susceptible individuals such as people who havecystic fibrosis, cancer, burns, diabetic ulcers or an immune systemdeficiency. In a hospital setting in particular, it has become resistantto many commonly used antibiotics.

According to data from the US Centers for Disease Control and Preventionand the National Nosocomial Infection Surveillance System, P. aeruginosais the second most common cause of nosocomial pneumonia, the third mostcommon cause of urinary tract infection, the fourth most common cause ofsurgical site infection, the seventh most frequently isolated pathogenfrom the bloodstream, and the fifth most common isolate overall from allsites (Solh and Alhajhusain, J Antimicrob Chemother. 64(2):229-38(2009)). Furthermore, P. aeruginosa is the most commonmultidrug-resistant (MDR) Gram-negative pathogen causing pneumonia inhospitalized patients (Goossens et al., Clin Microbiol Infect. 980-3(2003)).

Nonlimiting examples of infections caused by P. aeruginosa include: A)Nosocomial infections: 1. Respiratory tract infections especially incystic fibrosis patients and mechanically-ventilated patients; 2.Bacteraemia and sepsis; 3. Wound infections, particularly those of burnvictims; 4. Urinary tract infections; 5. Post-surgery infections oninvasive devises; 6. Endocarditis by intravenous administration ofcontaminated drug solutions; 7, Infections in patients with acquiredimmunodeficiency syndrome, cancer chemotherapy, steroid therapy,hematological malignancies, organ transplantation, renal replacementtherapy, and other conditions with severe neutropenia. B)Community-acquired infections: 1. Community-acquired respiratory tractinfections; 2. Meningitis; 3. Folliculitis and infections of the earcanal caused by contaminated water; 4. Malignant otitis externa in theelderly and diabetics; 5. Osteomyelitis of the caleaneus in children; 6.Eye infections commonly associated with contaminated contact lens; 7.Skin infections such as nail infections in people whose hands arefrequently exposed to water; 8. Gastrointestinal tract infections; and9. Muscoskeletal system infections.

In some embodiments, the lysin polypeptides of the present disclosureare used to treat a subject at risk for acquiring an infection due to P.aeruginosa and/or another Gram-negative bacterium. Subjects at risk foracquiring a P. aeruginosa or other Gram-negative bacterial infectioninclude for example, but are not limited to, cystic fibrosis patients,neutropenic patients, patients with necrotising enterocolitis, burnvictims, patients with wound infections, and more generally patients ina hospital setting, in particular surgical patients and patients beingtreated using an implantable medical device such as a catheter, forexample a central venous catheter, a Hickman device, orelectrophysiologic cardiac devices, for example pacemakers andimplantable defibrillators. Other patient groups at risk for infectionwith Gram-negative bacteria including P. aeruginosa include withoutlimitation patients with implanted prostheses such a total jointreplacement (for example total knee or hip replacement).

In one embodiment, the subject is suffering from a Gram-negativebacterial respiratory infection. In another embodiment, the subject issuffering from cystic fibrosis and each active ingredient isindependently administered in an inhalable composition, an oralcomposition or a buccal composition. In a more specific embodiment, thesubject is suffering from a Gram-negative bacterial respiratoryinfection associated with cystic fibrosis and each of the activeingredients is co-administered in an inhalable composition. In oneembodiment, the subject is suffering from a wound that has been infectedwith P. aeruginosa or another Gram-negative bacterium. An example of awound that is treatable by the methods of the present disclosure is aninfected burn or a burn at risk of becoming infected. Such burns includethermal (heat) burns, cold temperature burns, chemical burns, electricalburns, or radiation burns.

Additionally, P. aeruginosa and other Gram-negative bacteria frequentlycolonize hospital food, sinks, taps, mops, and respiratory equipment.The infection is spread from patient to patient via contact with fomitesor by ingestion of contaminated food and water (Barbara Iglewski,Medical Microbiology, 4th edition, Chapter 27, Pseudomonas, 1996).

In one embodiment, the lysin polypeptides of the present disclosure areused for the treatment of a Gram-negative bacterial infection (or of aninfection that has not been characterized) in a subject in combinationwith other therapies. Such optional combination therapy may compriseco-administering to the patient in need thereof an additionaltherapeutic agent, such as an antibiotic or other bactericidal orbacteriostatic agent, and/or another lysin targeting a differentcomponent of the pathogen's surface (for example, targeting a differentcomponent of the outer membrane). Besides antibiotics, bactericidal andbacteriostatic agents include but are not limited to lysins,disinfectatnts, antiseptics, and preservatives. Any of these can beoptionally used in combination with the lysin polypeptides of thepresent disclosure.

Antimicrobial disinfectants include, but are not limited tohypochlorites, chloramines, dichloroisocyanurate andtrichloroisocyanurate, wet chlorine, chlorine dioxide, peracetic acid,potassium persulfate, sodium perborate, sodium percarbonate and ureaperhydrate, iodpovidone, iodine tincture, iodinated nonionicsurfactants, ethanol, n-propanol and isopropanol and mixtures thereof;2-phenoxyethanol and 1- and 2-phenoxypropanol, cresols, hexachlorophene,triclosan, trichlorophenol, tribromophenol, pentachlorophenol, Dibromoland salts thereof, benzalkonium chloride, cetyl trimethylammoniumbromide or chloride, didecyldimethylammonium chloride, cetylpyridiniumchloride, berizethonium chloride, chlorhexidine, glucoprotamine,octenidine dihydrochloride, ozone and permanganate solutions, colloidalsilver, silver nitrate, mercury chloride, phenylmercury salts, copper,copper sulfate, copper oxide-chloride, phosphoric acid, nitric acid,sulfuric acid, amidosulfuric acid, toluenesulfonic acid, sodium,hydroxide, potassium hydroxide, and calcium hydroxide.

The combination of lysin polypeptides of the present disclosure withantiseptic reagents may provide more efficacy against Gram-negativebacteria than antibiotic combinations. Antiseptic reagents include, butare not limited to Daquin's solution, sodium or potassium hypochloritesolution, solution of sodium benzenesulfochloramide, certain iodinepreparations, such as iodopovidone, peroxides as urea perhydratesolutions and pH-buffered peracetic acid solutions, alcohols with orwithout antiseptic additives, weak organic acids such as sorbic acid,benzoic acid, lactic acid and salicylic acid, some phenolic compounds,such as hexachlorophene, triclosan and Dibromol, and cation-activecompounds, such as benzalkonium, chlorhexidine, methylisothiazolone,α-terpineol, thymol; chloroxylenol octenidine solutions.

The lysins of the present disclosure can be co-administered withstandard care antibiotics or with antibiotics of last resort,individually or in various combinations as within the skill of the art.Traditional antibiotics used against P. aeruginosa activity includeaminoglycosides, ticarcillin, ureidopenicillins, ceftazidime, cefepime,aztreonam, carbapenems, ciprofloxacin, levofloxacin, etc. (Table 2).Lysin polypeptides of the present disclosure may be co-administered withantibiotics used for the treatment of P. aeruginosa and others listed inTable 2. The list of antibiotics for other Gram-negative bacteria, suchas Klebsiella spp., Enterobacter spp., Escherichia coli, Citrobacterfreundii, Salmonella typhimurium, Yersinia pestis, and Franciscellatulerensis, will be similar as that provided above for P. aeruginosa andwill be standard care antibiotics for a particular bacterium involved oreven antibiotics of last resort (if the particular strain is resistantto standard care antibiotics).

Additional optional therapeutic agents to be coadministered include, butare not limited to the antibiotics mentioned above and those listed inTable 2, such as a ticarcillin-clavulanate combination,piperacillin-tazobactam combination, ceftazidime, cefepime,cefoperazone, ciprofloxacin, levofloxacin, imipenem, meropenem,doripenem, gentamicin, tobramycin, amikacin, polymyxin B, and colistin(polymixin E). For treatment of wounds, therapeutic agents to beco-administered include, but are not limited to, a propylene glycolhydrogel (e.g., SOLUGEL® (Johnson & Johnson)); an antiseptic; anantibiotic; and a corticosteroid.

TABLE 2 Antibiotics used for the treatment of Pseudomonas aeruginosaClass Agent Penicillins Ticarcillin-clavulanate Piperacillin-tazobactamCephalosporins Ceftazidime Cefepime Cefoperazone Monobactams AztreonamFlouroquinolones Ciprofloxacin Levofloxacin Carbapemens ImipenemMeropenem Doripenem Aminoglycosides Gentamicin Tobramycin AmikacinPolymixins Colistin Polymixin B

Antibiotic peptides such as polymyxin B and the related colistin(polymyxin E) have been used as antibacterial agents for the treatmentof P. aeruginosa bacterial infections. Thus, in one embodiment, thelysin polypeptides of the present disclosure are to be co-administeredwith polymyxin B and/or colistin.

Combining lysin polypeptides of the present disclosure with antibioticsprovides an efficacious new antimicrobial regimen. In one embodiment,co-administration of lysin polypeptides of the present disclosure withone or more antibiotics may be carried out at reduced doses and amountsof either the lysin or the antibiotic or both, and/or reduced frequencyand/or duration of treatment with augmented bactericidal andbacteriostatic activity, reduced risk of antibiotic resistance and withreduced risk of deleterious neurological or renal side effects (such asthose associated with colistin or polymyxin B use). Prior studies haveshown that total cumulative colistin dose is associated with kidneydamage, suggesting that decrease in dosage or shortening of treatmentduration using the combination therpy with lysin polypepides coulddecrease the incidence of nephrotoxicity (Spapen et al. Ann IntensiveCare. 1: 14 (2011)). As used herein the term “reduced dose” refers tothe dose of one active ingredient in the combination compared tomonotherapy with the same active ingredient. Ditto for “duration oftreatment.” In some embodiments, the dose of the lysin or the antibioticin a combination may be suboptimal or even subthreshold compared to therespective monotherapy.

In some embodiments, lysin polypeptides of the present disclosure areused to treat a bacterial infection such as an infection caused by adrug resistant Gram-negative bacteria. In further embodiments, lysinpolypeptides of the present disclosure alone or with one or moreantibiotics are used to treat bacterial infection such as an infectioncaused by a multi-drug resistant Gram-negative bacteria. Drug resistantor multi-drug resistant Gram-negative bacteria in the context of thisdisclosure inludes, but is not limited to P. aeruginosa.

In practice, infections are commonly polymicrobial, with mixedGram-positive and Gram-negative species (Citron et al. J Clin Microbiol.45(9): 2819-2828 (2007)). In some embodiments, lysin polypeptides of thepresent disclosure active against gram-negative bacteria can be used notonly with an antibiotic effective against gram-negative bacteria butalso in combination with one or more antibiotic and/or one or more otherlysins suitable for the treatment of Gram-positive bacteria depending onthe infection of a given subject.

In one embodiment, lysin polypeptides of the present disclosure arecapable of breaching or degrading a cell wall of Gram-negative bacteria.In a preferred embodiment, lysin polypeptides of the present disclosureare capable of breaching or degrading the cell wall of P. aeruginosa.

In some embodiments, the present disclosure provides a method ofinhibiting the growth of one or more Gram-negative bacteria comprisingadministering to a subject or delivering to a particular environment oneor more lysin polypeptides disclosed herein or a pharmaceuticallyacceptable composition thereof in an amount and under conditions suchthat the growth of Gram-negative bacteria is inhibited.

In another embodiment, the present disclosure provides a method ofinhibiting the growth of P. aeruginosa and/or one or more otherGram-negative bacteria comprising administering to a subject one or morelysin polypeptides disclosed herein in combination with other clinicallyrelevant agents.

In some embodiments, the present disclosure provides a method forincreasing the permeability of an outer membrane of P. aeruginosa and/orone or more other Gram-negative bacteria by contacting the outermembrane with (exposing the bacteria to) one or more lysin polypeptidesof the present disclosure.

In a further embodiment, the present disclosure provides a method forincreasing the permeability of an outer membrane of P. aeruginosa and/orone or more other Gram-negative bacteria by contacting the outermembrane with lysin polypeptides disclosed herein in combination withother clinically relevant agents, such as antibiotics, bactericidalagents, anticeptics, etc.

In some embodiments, the present disclosure provides a method ofaugmenting antibiotic activity of one or more antibiotics againstGram-negative bacteria compared to the activity of said antibiotics usedalone by administering to a subject one or more lysin polypeptidesdisclosed herein together with an antibiotic of interest. Thecombination is effective against bacteria and permits resistance againstthe antibiotic to be overcome and/or the antibiotic to be employed atlower doses, decreasing undesirable side effects, such as thenephrotoxic and neurotoxic effects of polymyxin B.

The compounds of the present disclosure can be used alone or incombination with additional permeabilizing agents of the outer membraneof the Gram-negative bacteria, including, but not limited to metalchelators as e.g. HDTA, TRIS, lactic acid, lactoferrin, polymyxins,citric acid (Vaara M. Microbiol Rev. 56(3):395-441 (1992)).

In one embodiment, the lysin polypeptides of the present disclosure arechemically modified. A chemical modification inludes but is not limitedto, adding chemical moieties, creating new bonds, and removing chemicalmoieties. Chemical modifications can occur anywhere in a lysinpolypeptide, including the amino acid side chains, as well as the aminoor carboxyl termini. Such modification can be present at more than onesite in a lysin polypeptide. Furthermore, one or more side groups, orterminal groups of a lysin polypeptide may be protected by protectivegroups known to the person ordinarily-skilled in the art.

In some embodiments, lysin polypeptides contain an attachment ofduration enhancing moieties. In one embodiment, the duration enhancingmoiety is polyethylene glycol. Polyethylene glycol (“PEG”) has been usedto obtain therapeutic polypeptides of enhanced duration (Zalipsky, S.,Bioconjugate Chemistry, 6:150-165 (1995); Mehvar, R., J. Pharm.Pharmaceut. Sci., 3:125-136 (2000)). The PEG backbone [(CH₂CH₂—O—)_(n),n: number of repeating monomers] is flexible and amphiphilic. Whenattached to another chemical entity, such as a lysin polypeptide, PEGpolymer chains can protect such lysin polypeptide from immune responseand other clearance mechanisms. As a result, pegylation can lead toimproved lysin polypeptide efficacy and safety by optimizingpharmacokinetics, increasing bioavailability, and decreasingimmunogenicity and dosing amount and/or frequency. “Pegylation” refersto conjugation of a PEG molecule with another compound, e.g. lysinpolypeptide.

In one embodiment, the present disclosure relates to the prevention,reduction, treatment, or removal of Gram-negative bacterialcontamination of medical devices, surfaces such as floors, stairs, wallsand countertops in hospitals and other health related or public usebuildings and surfaces of equipment in operating rooms, emergency rooms,hospital rooms, clinics, and bathrooms and the like. Examples of medicaldevices that can be protected using compositions described hereininclude but are not limited to tubings and other surface medicaldevices, such as urinary catheters, mucous, extraction catheters,suction catheters, umbilical cannulae, contact lenses, intrauterinedevices, intravaginal and intraintestinal devices, endotracheal tubes;bronchoscopes, dental prostheses and orthodontic devices, surgicalinstruments, dental instruments, tubings, dental water lines, fabrics,paper, indicator strips (e.g., paper indicator strips or plasticindicator strips), adhesives (e.g., hydrogel adhesives, hot-meltadhesives, or solvent-based adhesives), bandages, tissue dressings orhealing devices and occlusive patches, and any other surface devicesused in the medical field. The devices may include electrodes, externalprostheses, fixation tapes, compression bandages, and monitors ofvarious types. Medical devices can also include any device which can beplaced at the insertion or implantation site such as the skin near theinsertion or implantation site, and which can include at least onesurface which is susceptible to colonization by Gram-negative bacteria.

In one embodiment, the lysin polypeptides of the present disclosure areused for preserving food against Gram-negative bacterial contaminationcomprising adding to the food the compositions of the present disclosurecomprising lysin polypeptides. Examples of such food products are meatproducts (cured and/or uncured, fresh and/or cooked), salads and othervegetable products, drinks and dairy products, semi-processed foods,convenient foods as e.g. ready-to-eat meals and dried food products,etc.

One of the problems that bacteria pose towards humans is the formationof bio films. Biofilm formation occurs when microbial cells adhere toeach other and are embedded in a matrix of extracellular polymericsubstance (EPS) on a surface. The growth of microbes in such a protectedenvironment that is enriched with biomacromolecules (e.g.polysaccharides, nucleic acids and proteins) and nutrients allow forenhanced microbial cross-talk and increased virulence. As biofilm maydevelop in any supporting environment, a method or composition that canprevent or remove biofilm formation is needed. Pseudomonas aeruginosahas been shown to form biofilms on a variety of living and non-livingsurfaces such as the mucus plugs of the CF lung, contaminated catheters,contact lenses, etc (Sharma et al. Biologicals, 42(1):1-7 (2014)). Thus,in one embodiment, the lysin polypeptides of the present disclosure canbe used for prevention, control, disruption, and treatment of bacterialbiofilm, particulary those formed by or with the contribution of P.aeruginosa.

The lysin polypeptides of the present disclosure can be used in vivo,for example, to treat bacterial infections in a subject, as well as invitro, for example to treat cells (e.g., bacteria) in culture toeliminate or reduce the level of bacterial contamination of a cellculture.

Methods for Producing Lysin Polypeptides

In one embodiment, the present disclosure includes methods for producinglysin polypeptides of the present disclosure which kill or inhibit thegrowth of one or more Gram-negative bacteria, preferably P. aeruginosa,the method comprising culturing a host cell comprising a lysinpolynucleotide encoding one or more lysin polypeptides under suitableconditions to express the said polypeptide.

To obtain high level of lysin polypeptide expression, lysinpolynucleotide sequences are typically expressed by operatively linkingthem to an expression control sequence in an appropriate expressionvector and employing that expression vector to transform an appropriatecellular host. Such operative linking of a polynucleotide sequencesencoding lysin polypeptides of the present disclosure to an expressioncontrol sequence, includes, the provision of an initiation codon, ATG,in the correct reading frame upstream of the polynucleotide (DNA)sequence. Generally, any system or vector suitable to maintain,propagate or express polynucleotides and/or to express a polypeptide ina host may be used for expression of lysin-polypeptides. The appropriateDNA/polynucleotide sequence may be inserted into the expression systemby any of a variety of well-known and routine, techniques, such as, forexample, those set forth in Sambrook et al., Molecular Cloning, ALaboratory Manual. Additionally, tags can also be added to lysinpolypeptides to provide convenient methods of isolation, e.g., c-myc,biotin, poly-His; etc. Kits for such expression systems are commerciallyavailable.

A wide variety of host/expression vector combinations may be employed inexpressing the polynucleotide sequences encoding lysin polypeptides ofthe present disclosure. Large numbers of suitable vectors are known tothose of skill in the art, and are commercially available. Examples ofsuitable vectors are provided in Sambrook et al, eds., MolecularCloning: A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring HarborLaboratory (2001). Such vectors include, among others, chromosomal,episomal and virus-derived vectors, e.g., vectors derived from bacterialplasmids, from bacteriophage, from transposons, from yeast episomes,from insertion elements, from yeast chromosomal elements, from virusessuch as baculoviruses, papova viruses, such as SV40, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,and vectors derived from combinations thereof, such as those derivedfrom plasmid and bacteriophage genetic elements, such as cosmids andphagemids. Furthermore, said vectors may provide for the constitutive orinducible expression of lysin polypeptides of the present disclosure.More specifically, suitable vectors include but are not limited toderivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscolE1, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4,pBAD24 and pBAD-TOPO; phage DNAS, e.g., the numerous derivatives ofphage λ, e.g., NM989, and other phage DNA, e.g., M13 and filamentoussingle stranded phage DNA; yeast plasmids such as the 2 D plasmid orderivatives thereof; vectors, useful in eukaryotic cells, such asvectors useful in insect or mammalian cells; vectors derived fromcombinations of plasmids and phage DNAs, such as plasmids that have beenmodified to employ phage DNA or other expression control sequences; andthe like. Many of the vectors mentioned above are commercially availablefrom vendors such as New England Biolabs, Addgene, Clontech, LifeTechnologies etc. many of which also provide suitable host cells).

Additionally, vectors may comprise various regulatory elements(including promoter, ribosome binding site, terminator, enhancer,various cis-elements for controlling the expression level) wherein thevector is constructed in accordance with the host cell. Any of a widevariety of expression control sequences (sequences that control theexpression of a polynucleotide sequence operatively linked to it) may beused in these vectors to express the polynucleotide sequences encodinglysin polypeptides. Useful control sequences include, but are notlimited to: the early or late promoters of SV40, CMV, vaccinia, polyomaor adenovirus, the lac system, the tip system, the TAC system, the TRCsystem, the LTR system, the major operator and promoter regions of phageλ, the control regions of id coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase (e.g., Pho5), the promoters of the yeast-matingfactors, E. coli promoter for expression in bacteria, and other promotersequences known to control the expression of genes of prokaryotic oreukaryotic cells or their viruses, and various combinations thereof.

A wide variety of host cells are useful in expressing the lysinpolypeptides of present disclosure. Nonlimiting examples of host cellssuitable for expression of lysin polypeptides of the present disclosureinclude well known eukaryotic and prokaryotic hosts, such as strains ofE. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, andanimal cells, such as CHO, R1.1, B-W and L-M cells, African Green Monkeykidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells(e.g., Sf9), and human cells and plant cells in tissue culture. Whilethe expression host may be any known expression host cell, in apreferred embodiment the expression host is one of the strains of E.coli These include, but are not limited to commercially available E.coli strains such as Top10 (Thermo Fisher Scientific), DH5α (ThermoFisher Scientific), XL1-Blue (Agilent Technologies), SCS110(Stratagene), JM109 (Promega), LMG194 (ATCC), and BL21 (Thermo FisherScientific). There are several advantages of using E. coli as a hostsystem including: fast growth kinetics, where under the optimalenvironmental conditions, its doubling time is about 20 min (Sezonov etal., J. Bacteriol. 189 8746-8749 (2007), easily achieved high densitycultures, easy and fast transformation with exogenous DNA, etc. Detailsregarding protein expression in E. coli, including plasmid selection aswell as strain selection are discussed in details by Rosano, G. andCeccarelli, E., Front Microbiol., 5: 172 (2014).

Efficient expression of lysin polypeptides and vectors thereof dependson a variety of factors such as optimal expression signals (both at thelevel of transcription and translation), correct protein folding, andcell growth characteristics. Regarding, methods for constructing thevector and methods for transducing the constructed recombinant vectorinto the host cell, conventional methods known in the art can beutilized. While it is understood that not all vectors, expressioncontrol sequences, and hosts will function equally well to express thepolynucleotide sequences-encoding lysin peptides of the presentdisclosure, one skilled in the art will be able to select the propervectors, expression control sequences, and hosts without undueexperimentation to accomplish the desired expression without departingfrom the scope of this disclosure. In some embodiments, the presentinventors have found a correlation between level of expression andactivity of the expressed polypeptide; in E. coli expression systems inparticular, moderate levels of expression (for example between about 1and 10 mg/liter) have produced lysin polypeptides with higher levels ofactivity than those that were expressed at higher levels in in E. coli(for example between about 20 and about 100 mg/liter), the latter havingsometimes produced wholly inactive polypeptides.

Lysin polypeptides of the present disclosure can be recovered andpurified from recombinant cell cultures by well-known methods includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography, and lectin chromatography. Highperformance liquid chromatography can also employed for lysinpolypeptide purification.

Alternatively, the vector system used for the production oflysin-polypeptides of the present disclosure may be a cell freeexpression system. Various cell free expression systems are commerciallyavailable, including, but are not limited to those available fromPromega, LifeTechnologies, Clonetech, etc.

As mentioned above, there is an array of choices when it comes toprotein production and purification. Below, the inventors include by wayof nonlimiting example a general protocol that can be used for theproduction of lysin polypeptides of the present disclosure in E. coli.Examples of suitable methods and strategies to be considered in proteinproduction and purification are further provided in Structural GenomicsConsortium, Nat. Methods., 5(2): 135-146 (2008).

Exemplary Protocol:

1. DNA encoding lysin polypeptide is generated by total gene synthesis.

2. DNA fragments are next ligated into a preferably inducible vectorsuch as pBAD24 (inducible with arabinose) and transformed into E. colicells (for example TOP 10 from Invitrogen, Carlsbad, Calif.).

3. Transformed bacteria is spread on agar plates supplemented withbroth, such as Lysogeny broth (LB), vector induction agent (e.g., 0.2%arabinose) and a selsctable marker (e.g., 50 μg/ml carbenicillin) andincubated preferably overnight at 37° C.

4. Single close cultures of E. coli Top10 cells containing lysin plasmidare grown in broth supplemented with selectable marker (e.g., LBsupplemented with 50 μg/ml carbenicillin) preferably overnight at 37° C.The culture may then be diluted for example 1:200 in fresh mediumsupplemented with selectable marker (e.g., LB supplemented withcarbenicillin) and incubated for example for an additional 3 h. Lysinexpression is induced with addition of the inducible agent (e.g., 0.2%L-arabinose), and the cells are inubated preferably overnight at 30° C.

5. Cell pellet is resuspended in buffer (e.g., 20 mM Tris, pH 6.8) andhomogenized.

6. Protein solubilization and purification (using one or morechromatographic techniques) are performed in a well-buffered solutioncontaining a suitable ionic strength of a monovalent salt, e.g., anionic strength equivalent to 300-500 mM of NaCl.

7. Immobilized metal affinity chromatography (IMAC) is preferably usedas the initial purification step. If additional purification isrequired, size-exclusion chromatography (gel filtration) can be used ina further step. If necessary, ion exchange chromatography can be used asa final step.

Identification of Lysin Polypeptides

The present disclosure is based on identification of five lysins withpotent antibacterial activity against exponential phase Pseudomonasaeruginosa strain PAO1 (Examples 1 and 2). To identify the lysinpolypeptides of the present disclosure, the inventors used abioinformatics-based approach coupled with an antibacterial screen. Ofthe thus identified sequences, some were previously annotated asputative endolysins. However, the present inventors found that asubstantial majority among them (they screened over 80 polypeptides) didnot have any lysin activity or did not have activity against the targetorganism, P. aeruginosa. The five lysins identified as active weredesignated as GN37 (SEQ ID NO: 1), GN2 (SEQ ID NO: 2), GN4 (SEQ ID NO:3), GN14 (SEQ ID NO: 4), and GN43 (SEQ ID NO: 5). Initially, theinventors evaluated the ability of purified lysins (which weresynthesized, cloned into expression vector pBAD26, and then purified) topermeabilize the outer membrane (OM) of P. aeruginosa. (Example 1).

Most Gram-negative bacteria exclude hydrophobic compounds and do notallow the uptake of hydrophobic agents such as 1-N-phenylnaphthylamine(NPN), crystal violet, or 8-anilino-1-naphthalenesulfonic acid (ANS).The strong resistance to hydrophobic compounds is due to the presence ofouter membrane (OM), which contains associated proteins that anchor theOM to the peptidoglycan and keep it stable. Due to its hydrophobicnature, NPN fluoresces strongly under hydrophobic conditions and weaklyunder aqueous conditions (J Sokatch, The biology of Pseudomonas,December 2012, Elsevier). Accordingly, NPN fluorescence can be used as ameasurement of the outer membrane permeability.

In the present disclosure, the ability of numerous lysins (GN1, GN2,GN4, GN8, GN14, GN20, GN22, GN26, GN27, GN28, GN30, GN37, and GN43) topermeabilize the OM of P. aeruginosa strain PAO1 was tested byincubating NPN with PAO1 cells in the presence or absence of theabove-mentioned lysins. As shown in FIG. 3, incubation of NPN in thepresence of GN37 (SEQ ID NO: 1), GN2 (SEQ ID NO: 2), GN4 (SEQ ID NO: 3),GN14 (SEQ ID NO: 4), and GN43 (SEQ ID NO: 5) resulted in highestinduction of fluorescence compared with fluorescence emitted without thepresence of lysins (negative control). Moreover, each of the five lysins(GN2, GN4, GN14, GN37, and GN43) caused significantly stronger OMpermeability compared to that caused by the established permeabilizingagent EDTA (ethylene diamine tetraacetate). Furthermore, each of thefive lysins permeabilized the OM similarly to or better than a knownantibiotic of last resort used in the treatment of P. aeruginosa,Polymyxin B (PMB). The active lysins of the present disclosure generallyhave a C-terminal (except GN14 which has an N-terminal) alpha-helixamphipathic domain varying in size between 15 amino acid residues forGN14 and 33 amino acid residues for GN43. Common features of GN2, GN4,GN14, GN37, and GN43 including the sequence of the alpha-helicalamphipathic domain are included in Table 3. Secondary polypeptidestructure can be determined using various software programs such asJpred4 at www.compbio.dundee.ac.uk/jpred/. The amphipathic alpha helicesin particular were examined using Helical Wheel (kael.net/helical.htm).Nucleic acid sequences of GN37 (SEQ ID NO: 11), GN2 (SEQ ID NO: 12), GN4(SEQ ID NO: 13), GN14 (SEQ ID NO: 14), and GN43 (SEQ ID NO: 15) are alsoprovided (FIG. 1A and FIG. 2).

TABLE 3 General features of each lysin polypeptide andsequences of the corresponding C-terminal alpha-helical amphipathic domains (Bolded andunderlined region represents alpha-helical amphipathic domain). GN2hypothetical protein GOS_817346 [marine metagenome] GenBank: EDG23390.1147 amino acids, 16,790 Da, pI 6.1650-75% identical to range of Gram-negative phage lysinsMKISLEGLSLIKKFEGCKLEAYKCSAGVWTIGYGHTAGVKEGDVCTQEEAEKLLRGDIFKFEEYVQDSVKVDLDQSQFDALVAWTFNLGPGNLRSSTMLKKLNNGEYESVPFEMRRWNKAGG

(SEQ ID NO: 2) GN4 putative endolysin [Pseudomonas phage PAJU2]NCBI Reference Sequence: YP_002284361.1144 amino acids, 16,199 Da, pI 9.58 >90% identical to a range of P. aeruginosaphage lysozymes and muramidasesMRTSQRGIDLIKSFEGLRLSAYQDSVGVWTIGYGTTRGVTRYMTITVEQAERMLSNDIQRFEPELDRLAKVPLNQNQWDALMSFVYNLG AANLASSTLLKLLNKGDYQGAADQFP

EPLS (SEQ ID NO: 3) GN14 putative endolysin [Pseudomonas phage Lu11]NCBI Reference Sequence: YP_006382555.1189 amino acids, 20,380 Da, pI 9.14>97% identical to only three P. aeruginosa phage lysinsMNNELPWVAEARKYIGLREDTSKTSHNPKLLAMLDRMGEFSNES RAWWHDDETPWCGLFVGYCLGV

LTKLDRPAYGALVTFTRSGGGHVGFIVGKDARGNLMVLGGNQSNAVSIAPFAVSRVTGYFWPSFWRNKTAVKSVPFEERYSLPLLKSN GELSTNEA (SEQ ID NO: 3)GN37 peptidase M15 [Micavibrio aeruginasavorus]NCBI Reference Sequence: WP_014102102.1 126 amino acids, pI 9.69MTYTLSKRSLDNLKGVHPDLVAVVHRAIQLTPVDFAVIEGLRSVSRQKELVAAGASKTMNSRHLTGHAVDLAAYVNGIRW DWPLYDAIAVAVKAAAKELGVAIVWGGD

 (SEQ ID NO: 1) GN43 100% identical to MULTISPECIES: peptidase M23[Pseudomonas] NCBI Reference Sequence: WP_003085274.1439 amino acids, 48,311 da, pI 9.55MKRTTLNLELESNTDRLLQEKDDLLPQSVTNSSDEGTPFAQVEGASDDNTAEQDSDKPGASVADADTKPVDPEWKTITVASGDTLSTVFTKAGLSTSAMHDMLTSSKDAKRFTHLKVGQEVKLKLDPKGELQALRVKQSELETIGLDKTDKGYSFKREKAQIDLHTAYAHGRITSSLFVAGRNAGLPYNLVTSLSNIFGYDIDFALDLREGDEFDVIYEQHKVNGKQVATGNILAARFVNRGKTYTAVRYTNKQGNTSYYRADGSSMKKAFIRTPVDFARISSRFSLGRRHPILNKIRAHKGVDYAAPIGTPIKATGDGKILEAGRKGGYGNAVVIQHGQRYRTIYGHMSRFAKGIRAGTSVKQGQIIGYVGMTGLATGPHLHYEFQINGRHVDPLSAKL

(SEQ ID NO: 5)

Since GN2, GN4, GN14, GN37, and GN43 exhibited potentmembrane-permeabilizing activity, the antibacterial activity of each ofthe five lysins against P. aeruginosa strain PAO1 was evaluated (Example2). In addition to using EDTA and PMB as positive controls, humanlysozyme and novobiocin were included as well. Human lysozyme (HuLYS) isa naturally occurring antimicrobial peptide found in a variety oftissues, cells, and secretions involved in the pathophysiology of lunginfection (Callewaert et al. J. Biosci. 35:127-160 (2010)). It has beenshown to be effective against both Gram-positive and Gram-negativeorganisms, including P. aeruginosa by degrading peptidoglycans in thebacterial cell wall. Novobiocin (Albamycin, Cathamycin, Spheromycin) isan aminocoumarin antibiotic isolated from Streptomyces niveus. It ismostly active against Gram-positive bacteria, but certain Gram-negativestrains are also susceptible (Lindsey Grayson, Kucers' The Use ofAntibiotics: A Clinical Review of Antibacterial, Antifungal andAntiviral Drugs, CRC Press 6th Edition, 2010).

As shown in FIG. 4, all five lysins (GN2, GN4, GN14, GN37, and GN43)displayed greater antibacterial activity against P. aeruginosa strainPAO1 than either HuLYS or novobiocin alone, while GN4, GN14, and GN37exhibited equivalent antibacterial activity to that of EDTA and PMB.

GN37 was derived from Micavibrio aeruginosavorus, a predator of P.aeruginosa that has not-been previously used as a source ofanti-pseudomonas PGH activities. The use of live Micavibrioaeruginosavorus has been suggested as a biological-based agent tocontrol MDR P. aeruginosa (Dashiff et al. J Appl., 110(2):431-44(2011)). But to the inventors' knowledge there has been no report ofusing this organism as a source for individual antimicrobial proteins,PGHs, bacteriocins, antibiotics, etc. The inventors reasoned that anepibiotic predatory bacterium that attaches to the surface ofPseudomonas aeruginosa and extracts nutrients from within must encode ananti-pseudomonas PGH activity to pierce the outer membrane and cellwall. Based on this, the genomic sequence of Micavibrio aeruginosavorusstrain ARL-13 was scanned for genes annotated as PGH-like enzymes. Fivehydrolases were identified, cloned, and screened for anti-pseudomonasactivity. The locus now designatated as GN37 was the only ORF whichyielded a clearing zone (halo) on agar overlay plates. Thus, due to theunique source of GN37 and potent activity described in Example 3, GN37was examined in further detail. As illustrated in Example 3, a multiplesequence alignment comparing GN37 to various known or putative lysinsrevealed that GN37 is only 67% identical to Mitrecin A (Farris andSteinberg, Lett Appl Microbial., 58(5):493-502 (2014); and patentpublication US20140094401 A1). Unlike GN37, Mitrecin A was identified inthe genome of the Gram-positive organism (i.e., Streptomyces).Furthermore, the activity described for Mitrecin A is very weak comparedto that of GN37 (at roughly equivalent concentrations). Mitrecin A onlyachieved a <1-log decrease in bacterial viability over 16 hours ofincubation (against Yersinia pseudotuberculosis), compared to the >3-logdecrease after only 1 hour of GN37 treatment.

In addition to the full-length lysins disclosed here, the presentdisclosure provides peptide derivatives based on the C-terminalalpha-helical amphipathic domain of lysins of the present disclosure.Progressive truncation (amino acid deletion) of the C-terminal (and/orto of the N-terminal) of polypeptides comprising this domain can yieldactive lysin peptide fragments down to a minimum length active lysinpeptide. Such peptides can be further modified by addition of one ormore amino acids (other than those of the naturally occurring lysin) tothe truncated C- (or N-) terminal in a manner not disrupting the alphahelix. Both C- and N-terminal alpha-helical amphipathic domains can beidentified using bioinformatics approach. Examples of alpha helixnondisrupting amino acids are hydrophobic or charged residues thatextend the alpha helical region or that promote membrane insertion.Amino acid addition is further illustrated using the lysin polypeptideGN4 (Example 4, FIG. 5). While peptides FGN4-1 (SEQ ID NO: 7) and FGN4-2(SEQ ID NO: 8) are peptide fragments of GN4 (SEQ ID NO: 3), PGN4 (SEQ IDNO: 6), FGN4-3 (SEQ ID NO: 9), and FGN4-4 (SEQ ID NO: 10) each contain amodification (FIG. 5), a viability assay indicated that PGN4 and FGN4-3exhibit greater antibacterial activity than the other GN4 peptidestested (FIG. 6). PGN4-4 is a 39 amino acid polypeptide, which comprisesa 31 amino acid polypeptide FGN4-2 and an 8-residue antibacterialpeptide identified from the hepatitis B capsid (SQSRESQC) (amino acidnumbers 32-39 of SEQ ID NO: 6). Thus, as seen in FIG. 6, the addition ofSQSRESQC (amino acid numbers 32-39 of SEQ ID NO: 6) peptide augments theactivity of FGN4-2. Comparison of the native fragment FGN4-1 and FGN4-4,which has a C-terminus cysteine (FGN4-4) added, indicates that theaddition of cysteine to the C-terminus enhanced the activity of theFGN4-1. The cysteine was added to see if it would promote dimerizationand augment activity. The results indicate that the terminal cysteineaugments activity. Additional modifications include FGN4-2 in which 11C-terminal residues are removed. These residues are not required for thealpha helical structure (based on secondary protein structureconsiderations) and the inventors probed to see whether activity wouldbe maintained. Removal of all 11 residues did reduce activity butactivity could be restored and in fact improved by other modifications,such as those described earlier in this paragraph. In light of theforegoing, those of ordinary skill can readily produce truncated lysinswith their C-terminal alpha helical amphipathic domain intact byproducing lysin polypeptides progressively lacking one or more aminoacid residues from the C-terminal of this domain or from the N-terminalor both and testing such polypetides alone or in combination with one ormore antibiotics active against Gram-negative bacteria for activityagainst (i.e. ability to inhibit, reduce the population or kill) P.aeruginosa and/or another Gram-negative bacterium. Such testing canfollow for example the teachings provided in Examples 2, 3, 4, orProphetic Example 1. Of course, the testing procedures and protocolsthemselves are not limited to those in these Examples but can be anymethods known to those skilled in the art for assessing effectiveness ofan antibacterial and indeed an antimicrobial agent.

For the analysis of antibacterial activity in human serum, the GN lysinpolypeptides and GN peptides were each pooled in the presence andabsence of sub-MIC concentration of polymyxin B (Example 5, FIG. 7). PMBis a potent antibiotic with activity against P. aeruginosa and, at aconcentration of 1 mcg/ml, resulted in a <2-log 10 decrease in viabilityafter treatment for 1 hour in human serum. The GN4 peptide pool(containing each peptide at 25 mcg/ml, labeled “peptide pool” in FIG. 7)alone did not result in decreased viability and the lysin polypeptidepool (containing each GN lysin at a concentration of 25 mcg/ml) aloneresulted only in a <2-log 10 reduction in viability (FIG. 7). Whencombined with the PMB, however, both the peptide pool and the lysin poolresulted in a >4-log 10 decrease in viability (FIG. 7). These findingsindicate a strong additive or even synergistic effect of the combinationof PMB and the GN lysin polypeptides of the present disclosure. It isanticipated that individual lysin polypeptides will also result in asubstantial decrease in viability of Gram-negative bacteria such as P.aeruginosa if used individually instead of in a pool. An observationregarding FIG. 7 is that the peptide pool alone is not as active aswould have been predicted from FIG. 6. This is most likely due to thefact that biological activity of many antibacterial agents is diminishedin the presence of human serum (Zhanel et al., Antimicrob AgentsChemother. 42(9): 2427-2430 (1998)). However, once the lysin pool isco-administered with PMB, the antibacterial activity of the lysin poolis restored, despite of the presence of serum (FIG. 7). Thus, thedecrease in lysin pool antimicrobial activity in the presence of humanserum may be due to the antagonistic activity among the peptides in thepeptide pool that is no longer inhibitory in the presence of PMB.Alternatively, one or more proteins present in the human serum may haveantagonistic effect on the lysin pool, wherein the effect is repressedupon the addition of PMB. The two possible scenarios can bedistinguished by repeating the assay using individual lysinpolypeptides, performing the assay in serum in the presence or absenceof PMB, and comparing the results to those obtained using lysin pools.Furthermore, the inhibition of lysin pool activity in serum could be dueto the high salt concentrations disrupting the electrostatincinteraction between lysin and outer membrane.

The GN lysin polypeptides of the present disclosure have bacterialactivity distinct from traditional antibiotic, vaccine, and anti-toxintreatments, and are useful to combat infections caused by Gram-negativebacteria. As described in the Examples, unlike other treatments lysinsprovide a rapid bactericidal and, when used in sub MIC amountsbacteriostatic effect, and are active against a range of antibioticresistant bacteria, mirroring results previously obtained using specificlysins against Gram-positive bacteria including S. aureus, S. pyogenes,S. pneumoniae, B. anthracis, and B. cereits and have not been associatedwith evolving resistance (Fischetti, V. Curr Opin Microbiol., 11(5):393-400 (2008)). Based on the present disclosure, in a clinical setting,lysins are a potent alternative for treating infections arising fromdrug- and multidrug-resistant bacteria. Existing resistance mechanismsfor Gram-negative bacteria should not affect sensitivity to the PGHactivity of lysins.

The lysin polypeptides of the present disclosure differ from existingPGHs under development for the treatment of Gram-negative infections.Previously described artilysins consist of positively charged PGHs fusedto exogenously-derived cationic peptides (Briers et al., AntimicrobAgents Chemother. 58(7): 3774-84 (2014); Briers et al. MBio. 4:e01379-14(2014); U.S. Pat. No. 8,846,865). Moreover, the artilysins usepolycationic peptides that are not derived from endolysins. On thecontrary, polycationic regions of lysin polypetides of the presentdisclosure are derived from lysins that naturally interact with anddestabilize the OM of P. aeruginosa, a feature that is anticipated toresult in improved targeting efficiency against this pathogen.

The lysin polypeptides of the present disclosure need not be modified bythe addition of antimicrobial cationic peptides, although fusionpolypeptides containing such peptides added to lysin polypeptidesisolated and recombinantly generated as described herein are certainlybeing contemplated. However, even in the absence of added cationic orother antimicrobial (antibacterial) peptides, the lysin polypeptides ofthe present disclosure have substantial anti-Gram-negative antibacterialactivity, including both bacteriostatic and bactericidal activity. Theforegoing notwithstanding, the present disclosure also contemplatesfusion lysin polypeptides comprising a lysin polypeptide having nativeGram-negative antibacterial function fused to an antimicrobial peptide(AMP, defensin, sushi peptide, cationic peptide, polycationic peptide,amphipatic peptide, hydrophobic peptide) stretch such as those describedin US Patent Application US2015/0118731 and International PatentApplications WO 2014/0120074, WO 2015/070912; WO 2015/071436; WO2015/070911; WO 2015/071437; WO/2012/085259; WO 2014/001572, and WO2013/0344055. Fusion polypeptides containing additional bactericidalsegments, i.e., segments having bactericidal activity on their ownprior, to fusion or contributing positively to the bactericidal activityof the parent lysin polypeptide, are also contemplated.

Pharmaceutical Compositions and Preparations

The compositions of the present disclosure can take the form ofsolutions, suspensions, emulsion, tablets, pills, pellets, capsules,capsules containing liquids, powders, sustained-release formulations,suppositories, tampon applications emulsions, aerosols, sprays,suspensions, lozenges, troches, candies, injectants, chewing gums,ointments, smears, a time-release patches, a liquid absorbed wipes, andcombinations thereof.

Administration of the compositions of the present disclosure orpharmaceutically acceptable forms thereof may be topical, i.e., thepharmaceutical composition is applied directly where its action isdesired (for example directly to a wound), or systemic. In turn,systemic administration can be enteral or oral, i.e., substance is givenvia the digestive tract, parenteral, i.e., substance is given by otherroutes than the digestive tract such as by injection or inhalation.Thus, the lysin polypeptides of the present disclosure can beadministered to a subject orally, parenterally, by inhalation,topically, rectally, nasally, buccally or via an implanted reservoir orby any other known method. The lysin polypeptides of the presentdisclosure can also be administered by means of sustained release dosageforms.

For oral administration, the lysin polypeptides of the presentdisclosure can be formulated into solid or liquid preparations, forexample tablets, capsules, powders, solutions, suspensions anddispersions. The compound can be formulated with excipients such as,e.g., lactose, sucrose, corn starch, gelatin, potato starch, alginicacid and/or magnesium stearate.

For preparing solid compositions such as tablets and pills, a lysinpolypeptide of the present disclosure or a fragment thereof is mixedwith a pharmaceutical excipient to form a solid preformulationcomposition. If desired, tablets may be sugar coated or enteric coatedby standard techniques. The tablets or pills may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet of pill can include an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by enteric layer,which serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials, can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The topical compositions of the present disclosure may further comprisea pharmaceutically or physiologically acceptable carrier, such as adermatologically or anotically acceptable carrier. Such carriers, in thecase of dermatologically acceptable carriers, are preferably compatiblewith skin, nails, mucous membranes, tissues and/or hair, and can includeany conventionally used dermatological earner meeting theserequirements. In the case of otically acceptable carriers, the carrieris preferably compatible with all parts of the ear. Such carriers can bereadily selected by one of ordinary skill in the art. Carriers fortopical administration of the compounds of the present disclosureinclude, but are not limited to, mineral oil, liquid petroleum, whitepetroleum, propylene glycol, polyoxyethylene and/or polyoxypropylenecompounds, emulsifying wax, sorbitan monostearate, polysorbate 60, cetylesters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, andwater. In formulating skin ointments, the active components of thepresent disclosure may be formulated in an oleaginous hydrocarbon base,an anhydrous-absorption base, a water-in-oil absorption base, anoil-in-water water-removable, base and/or a water-soluble base. Informulating otic compositions, the active components of the presentdisclosure may be formulation in an aqueous polymeric suspensionincluding such carriers as dextrans, polyethylene glycols,polyvinylpyrrolidone, polysaccharide gels, Gelrite®, cellulosic polymerslike hydroxypropyl methylcellulose, and carboxy-containing polymers suchas polymers or copolymers of acrylic acid, as well as other polymericdemulcents. The topical compositions according to the present disclosuremay be in any form suitable for topical application, including aqueous,aqueous-alcoholic or oily solutions, lotion or serum dispersions,aqueous, anhydrous or oily gels, emulsions obtained by dispersion of afatty phase in an aqueous phase (OAV or oil in water) or, conversely,(W/O or water in oil), microemulsions or alternatively microcapsules,microparticles or lipid vesicle dispersions of ionic and/or nonionictype, creams, lotions, gels, foams (which will generally require apressurized canister, a sutiable applicator an emulsifier and an inertpropellant), essences, milks, suspensions, or patches. Topicalcompositions of the present disclosure may also contain adjuvants suchas hydrophilic or lipophilic gelling agents, hydrophilic or lipophilicactive agents, preserving agents, antioxidants, solvents, fragrances,fillers, sunscreens, odor-absorbers and dyestuffs. In a further aspect,the topical antibacterial compositions may be administered inconjunction with devices such as transdermal patches, dressings, pads,wraps, matrices and bandages capable of being adhered or otherwiseassociated with the skin or other tissue of a subject, being capable ofdelivering a therapeutically effective amount of one or moreantibacterial lysin polypeptides in accordance with the presentdisclosure.

In one embodiment, the topical compositions of the present disclosureadditionally comprise one or more components used to treat topicalburns. Such components typically include, but are not limited to, apropylene glycol hydrogel; a combination of a glycol, a cellulosederivative and a water soluble aluminum salt; an antiseptic; anantibiotic; and a corticosteroid. Humectants (such as solid or liquidwax esters), absorption prompters (such as hydrophilic clays, orstarches), viscocity building agents, and skin-protecting agents mayalso be added. Topical formulations may be in the form of rinses such asmouthwash. See, e.g., WO2004/004650.

The compounds of the present disclosure may also be administered byinjection of a therapeutic agent comprising the appropriate amount oflysin polypeptide and a carrier. For example, the lysin polypeptides canbe administered intramuscularly, intrathecally, subdermally,subcutaneously, or intravenously to treat infections by Gram-negativebacteria, more specifically those caused by P. aeruginosa. The carriermay be comprised of distilled water, a saline solution, albumin, aserum, or any combinations thereof. Additionally, pharmaceuticalcompositions of parenteral injections can comprise pharmaceuticallyacceptable aqueous or nonaqueous solutions of lysin polypeptides inaddition to one or more of the following: pH buffered solutions,adjuvants (e.g., preservatives, wetting agents, emulsifying agents, anddispersing agents), liposomal formulations, nanoparticles, dispersions,suspensions or emulsion,s as well as sterile powders for reconstitutioninto sterile injectable solutions or dispersions just prior to use.

In cases where parenteral injection is the chosen mode ofadministration, an isotonic formulation is preferably used. Generally,additives for isotonicity can include sodium chloride, dextrose,mannitol, sorbitol, and lactose. In some cases, isotonic solutions suchas phosphate buffered saline are preferred. Stabilizers can includegelatin and albumin. A vasoconstriction agent can be added to theformulation. The pharmaceutical preparations according to this type ofapplication are provided sterile and pyrogen free.

The diluent may further comprise one or more other excipient such as,e.g., ethanol, propylene glycol, an oil or a pharmaceutical acceptableemulsifier or surfactant.

In another embodiment, the compositions of the present disclosure areinhalable compositions. The inhalable compositions of the presentdisclosure can further comprise a pharmaceutically acceptable carrier.In one embodiment, lysin polypeptide(s) of the present disclosure areadvantageously formulated as a dry, inhalable powder. In specificembodiments, lysin polypeptides inhalation solution may further beformulated with a propellant for aerosol delivery. In certainembodiments, solutions may be nebulized.

A surfactant can be added to an inhalable pharmaceutical composition ofthe present disclosure in order to lower the surface and interfacialtension between the medicaments and the propellant. Where themedicaments, propellant and excipient are to form a suspension, asurfactant may or may not be required. Where the medicaments, propellantand excipient are to form a solution, a surfactant may or may not benecessary, depending in part, on the solubility of the particularmedicament and excipient. The surfactant may be any suitable, non-toxiccompound which is non-reactive with the medicament and whichsubstantially reduces the surface tension between the medicament, theexcipient and the propellant and/or acts as a valve lubricant.

Examples of suitable surfactants include, but are not limited to: oleicacid; sorbitan trioleate; cetyl pyridinium chloride; soya lecithin;polyoxyethylene(20) sorbitan monolaurate; polyoxyethylene (10) stearylether; polyoxyethylene (2) oleyl ether; polyoxypropylene-polyoxyethyleneethylene diamine block copolymers; polyoxyethylene(20) sorbitanmonostearate; polyoxyethylene(20) sorbitan monooleate;polyoxypropylene-polyoxyethylene block copolymers; castor oilethoxylate; and combinations thereof.

Examples of suitable propellants include, but are not limited to:dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane and carbon dioxide.

Examples of suitable excipients for use in inhalable compositionsinclude, but are not limited to: lactose, starch, propylene glycoldiesters of medium chain fatty acids; triglyceride esters of mediumchain fatty acids, short chains, or long chains, or any combinationthereof; perfluorodimethylcyclobutane; perfluorocyclobutane;polyethylene glycol; menthol; lauroglycol; diethylene glycolmonoethylether; polyglycolized glycerides of medium chain fatty acids;alcohols; eucalyptus oil; short chain fatty acids; and combinationsthereof.

In some embodiments, the compositions of the present disclosure comprisenasal applications. Nasal applications include for instance nasalsprays, nasal drops, nasal ointments, nasal washes, nasal injections,nasal packings, bronchial sprays and inhalers, or indirectly through useof throat lozenges, mouthwashes or gargles, or through the use ofointments applied to the nasal nares, or the face or any combination ofthese and similar methods of application.

In another embodiment, the pharmaceutical compositions of the presentdisclosure contain a complementary agent, including one or moreantimicrobial agents and or one or more conventional antibiotics. Inorder to accelerate the treatment of the infection, or augment theantibacterial effect, the therapeutic agent containing one or more lysinpolypeptides of the present disclosure may further include at least onecomplementary agent which can also potentiate the bactericidal activityof the lysin polypeptide. The complementary agent may be one or moreantibiotics used to treat Gram-negative bacteria. In preferredembodiment, the complementary agents is an antibiotic or antimicrobialagents used tor the treatment of infections caused by P. aeruginosa.

Dosage and Frequency of Administration to Subjects

The compositions of the present disclosure may be presented in unitdosage form and may be prepared by any methods well known in the art.The amount of active ingredients which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the duration of exposure of the recipient to theinfectious bacteria, the size and weight of the subject, and theparticular mode of administration. The amount of active ingredients thatcan be combined with a carrier material to produce a single dosage formwill generally be that amount of each compound which produces atherapeutic effect. Generally, out of one hundred percent, the totalamount will range from about 1 percent to about ninety-nine percent ofactive ingredients, preferably from about 5 percent to about 70 percent,most preferably from about 10 percent to about 30 percent.

Dosages administered depend on a number of factors including theactivity of infection being treated, the age, health and generalphysical condition of the subject to be treated, the activity of aparticular lysin polypeptide, the nature and activity of the antibioticif any with which a lysin polypeptide according to the presentdisclosure is being paired and the combined effect of such pairing.Generally, effective amounts of the present lysin polypeptides to beadministered are anticipated to fall within the range of 1-50 mg/kgadministered 1-4 times daily for a period up to 14 days. The antibioticif one is also used will be administered at standard dosing regimens orin lower amounts. All such dosages and regimens however (whether of thelysin polypeptide or any antibiotic administered in conjunctiontherewith) are subject to optimization. Optimal dosages can bedetermined by performing in vitro and in vivo pilot efficacy experimentsas is within the skill of the art, but taking the present disclosureinto account.

In some embodiments, time exposure to the active lysin polypeptide(s)units may influence the desired concentration of active lysinpolypeptide units per ml. Carriers that are classified as “long” or“slow” release carriers (such as, for example, certain nasal sprays orlozenges) could possess or provide a lower concentration of lysinpolypeptide units per ml, but over a longer period of time, whereas a“short” or “fast” release carrier (such as, for example, a gargle) couldpossess or provide a high concentration of lysin polypeptide units perml, but over a shorter period of time. There are circumstances where itmay be necessary to have a much higher unit/ml dosage or a lower unit/mldosage.

For any lysin polypeptide of the present disclosure, the therapeuticallyeffective dose can be estimated initially either in cell culture assaysor in animal models, usually mice, rabbits, dogs, or pigs. The animalmodel can also be used to achieve a desirable concentration range androute of administration. Obtained information can then be used todetermine the effective doses, as well as routes of administration inhumans. Dosage and administration can be further adjusted to providesufficient levels of the active ingridient or to maintain the desiredeffect. Additional factors which may be taken into account include theseverity of the disease state, age, weight and gender of the patient;diet, desired duration of treatment, method of administration, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy and the judgment of thetreating physician.

A treatment regimen can entail daily administration (e.g., once, twice,thrice, etc. daily), every other day (e.g., once, twice, three, etc.every other day), semi-weekly, weekly, once every two weeks; once amonth, etc. In one embodiment, treatment can be given as a continuousinfusion. Unit doses can be administered on multiple occasions.Intervals can also be irregular as indicated by monitoring clinicalsymptoms. Alternatively, the unit dose can be administered as asustained release formulation, in which case less frequentadministration is required. Dosage and frequency may vary depending onthe patient. It will be understood by one of skill in the art that suchguidelines will be adjusted for localized administration, e.g.intranasal, inhalation, rectal, etc., or for systemic administration,e.g. oral, rectal (e.g., via enema), i.m. (intramuscular), i.p.(intraperitoneal), i.v. (intravenous), s.c. (subcutaneous),transurethral, and the like.

EXAMPLES Example 1 Identification of Gram Negative Lysins

Putative PGH candidates, that may be used to kill Gram-negativebacteria, were identified using a bioinformatics search protocol. First,the inventors generated a short list of P. aeruginosa PGHs obtained fromannotated genome sequences that were screened with search terms forbacteriophage lysins, including “amidase”, “lysozyme”,“glucosaminidase”, “endopeptidase”, “peptidoglycan hydrolase”, “lytictransglycosylase”, “endolysin”, “lysin”, and “cell wall hydrolase”. ThePGHs identified in this manner were then used to search, by BLASTPanalysis, all P. aeruginosa genome sequences in GenBank (P. aeruginosagroup; Taxid: 136841) and the genomic sequence of Micavibrioaeruginosavorus strain ARL-13 to generate a larger group of putativePGHs. A subset of this group, comprised of 46 PGHs, was then chosen forfurther study—the criteria for inclusion here was diversity with respectto sequence conservation and including both highly and poorly conservedenzymes with a range of putative catalytic/cell wall binding activities.The 46 PGHs were synthesized, cloned into the bacterial expressionvector pBAD24 (Guzman et al., J Bacterial. (14):4121-30 (1995)), andtransformed into E. coli strain Top10 (Life Technologies). To assessactivity, all E. coli clones (including vector controls) were screenedusing a plate-based assay for lytic activity against P. aeruginosastrain PAO1. Positive clones were then further analysized with respectto the induction of soluble protein in liquid LB cultures in the mannerdescribed (Schuch et al., Nature, 418 (6900):884-9, (2002)). For thesoluble and active lysins, crude E. coli extracts of induced cultureswere then examined for the ability to induce permeabilizaton of P.aeruginosa strain PAO1 using the hydrophobic fluorescent probe1-N-phenylnaphtthylmine (NPN). The NPN assay is a standard method(Helander and Mattila-Sandholm, J Appl Microbiol., 88(2):213-9, (2000))to screen for compounds that disrupt the bacterial outer membrane. Thisscreening approach ultimately yielded 5 candidate PGHs (with N- andC-terimnal alpha helical domains) in the genomes of Pseudomonasaeruginosa strains, and other Gram-negative organisms (includingMicavibrio aeruginosavorus), as well as those identified using marinemetagenomics. The resulting proteins were then tested for theirantibacterial activity against P. aeruginosa strain PAO1, which isresistant to penem antibiotics (Okamoto et al, Antimicrob AgentsChemother. 45(7):1964-71 (2001)).

In order to evaluate the activity of purified lysins, the uptake ofhydrophobic fluorescent probe 1-N-phenylnaphtthylmine (NPN) by P.aeruginosa strain PAO1 was examined. Since the outer membrane ofGram-negative bacteria acts as a permeability barrier to hydrophobiccompounds, including lysins and NPN, the permeabilizing activity of GNscan be and was evaluated by the ability of hydrophobic compound to reachthe inner target. Thus, while NPN is normally excluded by the outermembrane, it exhibits prominent fluorescence intensity when itpartitions into the outer membrane lipid bilayer.

P. aeruginosa PAO1 was obtained from the American type CultureCollection (ATCC). Bacteria were cultured at initial concentration of10⁵ CFU/ml in LB medium (Sigma-Aldrich) in a shaker-incubator at 37° C.and 250 r.p.m. P. aeruginosa culture was grown to the beginning ofexponential phase (A550˜0.3) to which each of the candidate lysins (GNs)was added at a concentration of 10 mcg/ml. To measure the uptake of NPN,10 μM NPN was added to exponentially growing cells containing GN1, GN2,GN4, GN8, GN14, GN20, GN22, GN26, GN27, GN28, GN30, GN37, and GN43 inPBS and fluorescence was monitored at 420 nm at 1 hour with afluorescence spectrophotometer (FIG. 3). Antibiotic polymyxin B (5mcg/ml) and EDTA (1 mM) were used as positive controls, while cellstreated with NPN but without the addition of lysin served as a negativecontrol.

As shown in FIG. 3, a strong fluorescent signal was observed when P.aeruginosa cells were treated with GN2 (SEQ ID NO: 2), GN4 (SEQ ID NO:3), GN14 (SEQ ID NO: 4), GN37 (SEQ ID NO: 1), or GN43 (SEQ ID NO: 5) inthe presence of NPN (grey bars). The general features of each lysin areshown in Table 4.

Collectively, these results identify a group of GN lysins that exhibitan ability to permeate the OM of Gram-negative bacteria, establishing afirst step in identifying lysins active against Gram-negative bacteriawithout the need to resort to addition of heterologous peptide segments.

TABLE 4 General Features of G2, GN4, GN14, GN37, and GN43. GenBank LysinSize (aa) pI Class Source Accession Homology Feature GN2 147 6.16Lysozyme Marine EDG23390.1 50-75% range of α-helical metagenomeGram-lysins C-terminus GN4 144 9.58 Lysozyme PseudomonasYP_002284361.1 >90% (Pa phage α-helical phage PAJU2 lysins) C-terminusGN14 189 9.14 NLPC_P60 Pseudomonas YP_006382555.1 >97% to 3 Pa Amidase?phage PAJU2 phage lysins GN37 126 9.69 Peptidase_M15_4 MicavibrioWP_014102102.1 ≤67% to bacteriocin Large α-helical aeruginosavorus andG-petidases N-terminal binding-domain GN43 439 9.55 LysM-Peptidase_M 23Pseudomonas PaerPAb_03459 ≤100% to range Multi Spp. of pseudomonasdomain peptidases

Example 2 GN Lysins Exhibit Strong Antibacterial Activity AgainstGram-Negative Bacteria

In order to evaluate the antibacterial activity of purified lysins, theviability of live P. aeruginosa strain PAO1 following the incubationwith individual GN lysins was evaluated. Briefly, 10⁶ PAO1 cells weretreated with the indicated GN lysin at a concentration of 25 mcg/ml for1 hour (at 37° C., without agitation) in the presence of 20 mM Tris-HCl(pH 7.2) buffer. Polymyxin B (PMB, at 25 mcg/ml), novobiocin (Nov, at 5mcg/ml), EDTA (1 mM) and human lysozyme (HuLYZ, at 25 mcg/ml) were usedas controls. The threshold of detection was 2.0 Log 10 CFU/ml.

As FIG. 4 shows, each GN lysin exhibited greater antibacterial activitythan either HuLYZ or novobiocin, while each of GN4 (SEQ ID NO: 3), GN14(SEQ ID NO: 4), and GN37 (SEQ ID NO: 1) showed stronger or equivalentantibacterial activity when compared to EDTA and PMB respectively.

This experiment demonstrates that GNs can exhibit equivalent or greaterantibacterial activity than any of conventional antibiotics, humanlysozyme or the chelating agent EDTA against Gram-negative bacteria.

Example 3 GN37 is Highly Effective Gram-Negative Antibacterial Agent

GN37 lysin is a 126 amino acid polypeptide (FIG. 1A) encoded by the381-bp MICA_542 locus of Micavibrio aeruginosavorus. M. aeruginosavorusis a predator of P. aeruginosa and has not been previously exploited asa source of anti-pseudomonas PGH activities. The GN37 lysin is a highlypositively charged protein with a predicted pI of 9.69. Additionally,GN37 is a member of the Peptidase_M15_4 family of PGHs with DD- andDL-carboxypeptidase activities (including members of the VanY superfamily) (FIG. 1B). Based on BLASTP analysis, GN37 (SEQ ID NO: 1) is ≤67%[identical to proteins from >50 different Gram-negative species and 1Gram-positive bacterium species. A multiple sequence alignment is shownin FIG. 1C comparing GN37 to the Gram-positive homolog (fromStreptomyces, GenBank sequence AGJ50592.1) and proteins fromGram-negative pathogens including E. coli (WP_001117823.1 andNP_543082.1), Yersinia spp. (CAJ28446.1) and Acinetobacter baumannii,(WP_034684053.1). Importantly, there are no sequences in the publicdatabase with >67% identity to GN37.

Example 4 Peptide Derivatives of GN4 Exhibit Potent AntibacterialActivity

In addition to the full-length lysins, five peptide derivatives of GN4(corresponding to an alpha-based helical C-terminal fragment) were alsogenerated and examined (FIG. 5). The first peptide (FGN4-1, (SEQ ID NO:7)) corresponds to a 42 amino acid C-terminal alpha-helical domain. Thiswas arrived at taking into account protein secondary structurepredictions that this region was sufficient to generate the C-terminalalpha-helical amphipathic domain of GN4 (alternatively it could havebeen armed at by progressive truncation of the very end of theC-terminal and testing the effect on activity each time). The rationalefor the additional modifications has been described elsewhere in thespecification

A single C-terminal cysteine was added to FGN4-1 (SEQ ID NO: 7) togenerate FGN4-4 (SEQ ID NO: 10). For the 3 additional peptides, the 11C-terminal residues of FGN4-1 were either removed (FGN4-2, (SEQ ID NO:8)) or removed and replaced with either a single lysine (K) residue(FGN4-3, (SEQ ID NO: 9)) or an 8-residue antibacterial peptideidentified from the hepatitis B capsid (PGN4, (SEQ ID NO: 6)). Thehepatitis B peptide is SQSRESQC (amino acid numbers 32-39 of SEQ ID NO:6) and has Epitope ID number 96916 from the Immune Epitope Database. Theantibacterial activity of each GN4-derived peptide was examined in akilling assay (FIG. 6), where 10⁵ PAO1 cells were treated with theindicated peptide derivative at a concentration of 10 mcg/ml for 1 hour(at 37° C., without agitation) in the presence of 20 mM Tris-HCl (pH7.2) buffer. The threshold of detection was 2.0 log 10 CFU/ml. Asillustrated in FIG. 6, each of the tested peptides showed someantibacterial activity (compared to the buffer that served as a negativecontrol), whereas FGN4-1 and FGN4-4 exhibited superior activity, witha >4 log decrease in cell viability.

Example 5 GN Lysins and GN Peptides Display Robust AntibacterialProperties

The antibacterial properties of GN lysins and GN-peptides were nextassessed in human serum. P. aeruginosa cells were incubated in humanserum for 1 h at 37° C., without agitation, and the activity againstexponential phase of PAO1 was examined. GN Lysins and GN peptides (at 25mcg/ml) were each pooled in the presence and absence of a sub-MICconcentration of polymyxin B (PMB) (1 mcg/ml). The threshold ofdetection was 2.0 log 10 CFU/ml. As shown in FIG. 7, GN lysin pool alonedemonstrated antibacterial properties similar to those displayed byPolymxin B. Furthermore, when combined with PMB, both the peptide pooland the lysin pool resulted in a ≥4-log 10 decrease in viability.

Collectively, these findings indicate a strong additive or synergisticactivity between PMB and either the lysin polypeptides (GNs) or lysinpeptides in human serum. Since prior studies have demonstrated strongantimicrobial activity of individual components of a combined lysinmixture, it is anticipated that in addition to lysin pools, individuallysin components will also exhibit a strong additive or synergisticactivity with antibiotics (Loeffler et al. Antimicrob Agents Chemother.January; 47(1): 375-377 (2003)).

Prophetic Example 1 Testing of Isolated GN Lysins

In this Example, the goal is to verify that GN lysin polypeptides andpeptide derivatives of GN4 described herein are individually capable ofinhibiting the growth of or killing Gram-negative bacterial strains inaddition to P. aeruginosa. To do so, the isolated polypeptides of thisdisclosure (which may be expressed as described herein and purified byconventional techniques) will be tested for antimicrobial, activityagainst various Gram-negative strains such as Klebsiella pneumoniae NCTC9633 (ATCC 13883), Enterobacter aerogenes NCTC 10006 (ATCC 13048),Citrobacter freundii NCT 9750 (ATCC 8090), Salmonella typhimurium CDC6516-60 (ATCC 14028), Yersinia pestis (ATCC BAA-1511D-5), Francisellatulerensis (ATCC 6223) and Escherichia coli DSM 1103 (ATCC 25822), allcommercially available. In additional arms of this experiment, thestrain can be selected to be resistant to one or more standard careantibiotics, such as multi-drug resistant P. aeruginosa Br667strain.

Briefly, GN lysin polypeptides and GN4 peptides will be tested at aconcentration of 5-15 mcg/ml for 1 hour (at 37° C., without agitation)in the presence of 20 mM Tris-HCl (pH 7.2) buffer. The threshold ofdetection will be 2.0 Log 10 CFU/ml. The activity of each lysinpolypeptide or fragment thereof will be tested alone, or in combinationwith an antibiotic active against Gram-negative bacteria such aspolymyxin B or another antibiotic active against Gram-negative bacteriasuch as gentamicin. In arms of this experiment wherein the ability of alysin polypeptide to overcome resistance to an antibiotic is beingtested, the selected antibiotic will be one to which the particularbacterium is resistant. A 100 μL aliquot of inoculum will be removedfrom the culture medium at different time-points (0, 1, 4 and 8 h) forthe determination of CFU/mL by the plate count technique.

Based on the conservation of the outer membrane structure ofGram-negative bacteria, and on the effectiveness of GN4 and itsforegoing derivatives and other lysins of the present disclosure andExamples against P. aeruginosa, it is anticipated that the lysinpolypeptides of the present disclosure will exhibit antibacterialactivity against additional (other than P. aeruginosa) strains ofGram-negative bacteria.

Prophetic Example 2 Testing the Synergistic and Additive Effects BetweenGN Lysins/Lysin Peptides and Additional Antibiotics

Similar to the experimental design described in Example 5, the lysinpolypeptides disclosed herein will be tested for synergistic or additiveeffects when used in combination with additional (other than PMB)antibiotics. The synergistic or additive antibacterial properties of GNlysins and GN-peptides will be assessed in human serum. P. aeruginosawill be incubated in human serum for 1 h at 37° C., without agitation,and the activity against exponential phase of PAO1 will be examined. GNLysins and GN peptides (at 10.25 mcg/ml) will each be incubated in thepresence and absence of a sub-MIC concentration of an antibiotic such asnovobiocin, an aminoglycoside, Carbapenem, Ceftazidime (3rd generation),Cefepime (4th generation), Ceftobiprole (5th generation), aFluoroquinolone, Piperacillin, Ticarcillin, Colistin, a Rifamycin (suchas rifampicin, rifabutin, rifapentin etc.), and a Penicillin. Bacterialviability will be determined in CFU/mL by the plate count technique.

Prophetic Example 3 Testing of Lysin Polypeptides for the Treatment ofMultidrug-Resistant P. aeruginosa Using In Vivo Model of Pneumonia

Adult C57 Black mice will be maintained in a controlled environment incages in specific pathogen-free conditions. They will be brieflyanesthetized with inhaled sevorane (Abbot Laboratories) in an oxygenatedchamber and placed in a supine position with their heads elevated. MDRP. aeruginosa strain Ka02 bacterial inoculums (10⁶ cfu's in 50 μl oflactated Ringer's solution) will be instilled slowly into the left lungof each animal using a gavage needle.

Animals will be randomly separated into 3 experimental treatment groups.The 3 groups will consist of the following: 1) Saline-Treated Group(n=5), wherein saline will be administered i.v. 1 hr after infectionfollowed by one further i.p. administration at 5 hr post-infection; 2)Lysin Polypeptide GN4 Group (n=5), wherein GN4 will be administered i.v.1 hr after infection at a dose of 20 mg/kg followed by one further i.p.administration at 5 hr post-infection at a dose of 15 mg/kg; and 3)Positive control-Imipenem-Treated Group (n=5), wherein “Tienam” (Merck,Sharpe and Dohme)] (active ingredient, imipenem, a carbapenemantibiotic) will be administered i.p. 1 hr after infection at a dose of25 mg/kg followed by further i.p. administrations at 25 mg/kg at timepoints of 5 hr, 24 hr, 29 hr, 48 hr and 53 hr following infection.

Survival will be monitored for all three groups every 12 hrs until day 9post-infection. The same experimental design will be used to testantimicrobial activity in vivo of additional lysin polypeptidesdisclosed herein.

Prophetic Example 4 Testing of Protection Potential of LysinPolypeptides Against Pseudomonas aeruginosa Induced Sepsis in a MouseModel of Neutropenia

The protective efficacy of the lysin polypeptides against invasiveinfection with Pseudomonas aeruginosa will be measured in theneutropenic mouse model, described previously (Pier et al. Infect.Immun. 57:174-179 (1989); Schreiber et al. J. Immunol. 146:188-193(1991)). Adult BALB/c ByJ mice (Jackson Laboratories, Bar Harbor, Me.)will be maintained in a pathogen-free, pseudomonas-free environment.Neutropenia will be established by administering 3 mg ofcyclophosphamide (Cytoxan®, Bristol-Myers Squibb, Princeton, N.J.)intra-peritoneally to each mouse on days 1, 3, and 5. On day 5, thecyclophosphamide will be administered at time 0 hours, and 2 hourslater, lysin (at 20 mg/kg) or PBS control will be administered ip,followed by 10³ cfu of live Pseudomonas aeruginosa 06ad PA two hourslater. Mice will be monitored daily thereafter and mortality will bemeasured as an outcome. Furthermore, in order to determine optimal lysinconcentration for the treatment of animals, additional concentrations oflysin polypeptides will be tested.

The same experimental design will be used to test the protectiveantimicrobial activity in vivo of additional lysin polypeptidesdisclosed herein.

Prophetic Example 5 Treatment of Systemic Infection in a Human Subject

A patient diagnosed with systemic respiratory infection due to themultidrug resistant P. aeruginosa will be treated with intravenous oraerosolized polymyxin B, in combination with one or more lysinpolypeptides disclosed herein.

The recommended dosage of intravenous polymyxin B is 1.5 to 2.5mg/kg/day (1 mg=10,000 IU) (Sobieszczyk M E et al., J AntimicrobChemother., 54(2):566-9 (2004)). IV infusions of polymyxin B will begiven over 60-90 minutes for 15 days. A pharmaceutical compositioncomprising one or more lysin polypetides of the present disclosure willbe administered intravenously daily at a predetermined concentration.Alternatively, aerosolized polymyxin B therapy will be administereddaily (for 14 days) at 2.5 mg/kg/day in combination with apharmaceutical composition comprising one or more lysin polypetides ofthe present disclosure. Depending on a clinical status of an individualpatient, duration and/or concentration of treatment regiment may vary.Additionally, intravenous or aerosolized polymyxin B can be administeredin multiple occurrences during the day. Detailed information regardingthe use of intravenous and aerosolized polymyxins is provided, e.g., byFalagas et al. Clin Med Res. 4(2):138-146 (2006)).

At the end of the treatment, patients will be evaluated formicrobiological clearance as well as for safety. It is anticipated thattreatment of patients affliccted with systemic respiratory infectionwill lead to reduction of symptoms associated with the infection and/ora decrease or eradication of the causative bacterial population in thepatients.

Prophetic Example 6 Topical Treatment of Diabetic Ulcer in a HumanSubject

A diabetic foot ulcer infection is one of the severe complications ofdiabetes mellitus and one of the leading causes of hospitalization amongdiabetic patients. Importantly, P. aeruginosa frequently causes severetissue damage in diabetic foot ulcers (Sivanmaliappan and Sevanan, Int JMicrobiol. article ID: 605195 (2011). In order to show efficacy of lysinpolypetides of the present disclosure in the treatment of diabetic footulcer infection, a clinical study will be conducted.

Initially, a wound culture specimen of an individual patient will betested for the presence of P. aeruginosa. Next, susceptibility of P.aeruginosa to one or more lysin polypeptide(s) of the present disclosure(e.g., in a pool) will be confirmed using for example the disc diffusionmethod according to the Clinical and Laboratory Standards Institute(CLSI) criteria (van der Heijden et al. Ann Clin Microbiol Antimicrob.6:8 (2007)). Alternatively, the patients will be pre-selected to fulfillthe microbial susceptibility to lysin criterion. In parallel, thesusceptibility of an individual strain to a suitable antibiotic to beused, e.g., polymyxin B will be determined, in vitro (or the patientswill be preselected to fulfill the criterion of microbial susceptibilityto a specific antibiotic). A susceptible strain, e.g., P. aeruginosaATCC 27853 (susceptible to polymyxin) may be used as quality control(QC). In addition, the susceptibility of P. aeruginosa present in awound culture specimen to a combination therapy comprising both lysinpolypeptide(s) and an antibiotic of choice, such as polymyxin B (toexclude the possibility of lysin inhibition by the wound environment—seeExample 5 and FIG. 7 and discussion thereof above) will be performed.

Following in vitro susceptibility studies, the inventors will test invivo efficacy of lysin polypeptides of the present disclosure in thetreatment of diabetic ulcer infections. Lysin may be administered byinjection, e.g., intravenous or subcutaneous injection or can be applieddirectly to the wound as a topical formulation. Furthermore, lysinpolypeptides will be tested in combination with an antibiotic used forthe treatment of diabetic ulcer, wherein the antibiotic may beadministered systemically (orally or parenterally) or topically asindicated according to the standard dosage of a specific antibiotic. Forexample, if polymyxin B is used in combination with lysin polypeptides,the recommended dosage of intravenous polymyxin B is 1.5 to 2.5mg/kg/day.

Since diabetic foot ulcer infections are commonly polymicrobial, withmixed Gram-positive and Gram-negative species (Citron et al. J ClinMicrobiol. 45(9): 2819-2828 (2007)), lysin polypeptides of the presentdisclosure can be used in combination with more than one antibioticand/or one or more other lysins suitable for the treatment ofGram-positive bacteria present in the foot ulcer infection of a givenpatient. It is anticipated that lysin treatment will be followed by ahigh response rate for example in terms of log reduction or eradicationof bacterial population or any other clinical or laboratory measure ofimprovement. The response to a combination of a lysin according to thepresent disclosure and an antibiotic is anticipated to be even higher aslysins and antibiotics synergize. Indeed it may be possible to reducethe administered amount of one or both of a lysin or an antibioticwithout sacrificing effectiveness.

Prophetic Example 7 Topical Treatment of Burn in a Human Subject

Burn wound infections pose significant concerns as they delay healing,promote scarring and may result in bacteremia, sepsis or organ failure.P. aeruginosa is the most common source of burn infections (Church etal. Clinical Microbiology Reviews, 19 (2), 403-434 (2006)). Becauselysin polypeptides of the present disclosure have been shown to beactive against P. aeruginosa, it is anticipated that they can be usedfor the treatment of such infections as monotherapy or advantageously incombination without one or more antibiotics.

Topical compositions in a form of cream, gel, and/or foam comprisinglysin polypeptides of the present disclosure will be applied on theaffected skin area of patients suffering from various degree burns,including I, II, and III degree burns. The topical compositioncontaining lysin polypeptides with or without antibiotics or additionalactive agents (lysins active against different target organisms andoptionally antibiotics addressing these target organisms) will beapplied directly to/the infected burn area for different time intervals.

Topical application of lysin polypeptides is anticipated to result inthe reduction or elimination of patients' symptoms, as well as in thereduction or eradication of the causative bacterial population.

The foregoing Examples are illustrative of the methods and featuresdescribed herein and are not intended to be limiting. Moreover, theycontain statements of general applicability to the present disclosureand such statements are not confined to the particular Example theyappear in but constitute conclusions descriptions and expressions ofbroader implication of the experimental results described herein.

The contents of all cited references are incorporated by reference intheir entirety as if fully transcribed herein for all purposes.

SEQ ID NO: 1 GN37 Polypeptide sequendeMTYTLSKRSLDNLKGVHPDLVAVVHRAIQLTPVDFAVIEGLRSVSRQKELVAAGASKTMNSRHLTGHAVDLAAYVNGIRWDWPLYDAIAVAVKAAAKELGVAIVWGGDWTTFKDGPHFELDRSKYR (SEQ ID NO: 1) SEQ ID NO: 2 GN2Polypeptide sequence MKISLEGLSLIKKFEGCKLEAYKCSAGVWTIGYGHTAGVKEGDVCTQEEAEKLLRGDIFKFEEYVQDSVKVDLDQSQFDALVAWTFNLGPGNLRSSTMLKKLNNGEYESVPFEMRRWNKAGGKTLDGLIRRRQAESLLFESKEWHQV (SEQ ID NO: 2) GN4Polypeptide sequence SEQ ID NO: 3MRTSQRGIDLIKSFEGLRLSAYQDSVGVWTIGYGTTRGVTRYMTITVEQAERMLSNDIQRFEPELDRLAKVPLNQNQWDALMSFVYNLGAANLASSTLLKLLNKGDYQGAADQFPRWVNAGGKRLDGLVKRRAAERALFLEPLS (SEQ ID NO: 3) GN14Polypeptide sequence SEQ ID NO: 4MNNELPWVAEARKYIGLREDISKTSHNPKLLAMLDRMGEFSNESRAWWHDDETPWCGLFVGYCLGVAGRYVVREWYRARAWEAPQLTKLDRPAYGALVTFTRSGGGHVGFIVGKDARGNLMVLGGNQSNAVSIAPFAVSRVTGYFWPSFWRNKTAVKSVPFEERYSLPLLKSNGELSTNEA (SEQ ID NO: 4) GN43 Polypeptide sequenceSEQ ID NO: 5 MKRTTLNLELESNTDRLLQEKDDLLPQSVTNSSDEGTPFAQVEGASDDNTAEQDSDKPGASVADADTKPVDPEWKTITVASGDTLSTVFTKAGLSTSAMHDMLTSSKDAKRFTHLKVGQEVKLKLDPKGELQALRVKQSELETIGLDKTDKGYSFKREKAQIDLHTAYAHGRITSSLFVAGRNAGLPYNLVTSLSNIFGYDIDFALDLREGDEFDVIYEQHKVNGKQVATGNILAARFVNRGKTYTAVRYTNKQGNTSYYRADGSSMRKAFIRTPVDFARISSRFSLGRRHPILNKIRAHKGVDYAAPIGTPIKATGDGKILEAGRKGGYGNAVVIQHGQRYRTIYGNMSRFAKGIRAGTSVKQGQIIGYVGMTGLATGPHLHYEFQINGRHVDPLSAKLPMADPLGGADRKRFMAQTQPMIARMDQEKKTLLALNKQR (SEQ ID NO: 5) PGN4Polypeptide sequence SEQ ID NO: 6NKGDYQGAADQFPRWVNAGGKRLDGLVKRRASQSRESQC (SEQ ID NO: 6) FGN4-1Polypeptide Sequence SEQ ID NO: 7NKGDYQGAADQFPRWVNAGGKRLDGLVKRRAAERALFLEPLS (SEQ ID NO: 7) FGN4-2Polypeptide Sequence SEQ ID NO: 8NKGDYQGAADQFPRWVNAGGKRLDGLVKRRA (SEQ ID NO: 8) FGN4-3Polypeptide sequence SEQ ID NO: 9NKGDYQGAADQFPRWVNAGGKRLDGLVKRRK (SEQ ID NO: 9) FGN4-4Polypeptide sequence SEQ ID NO: 10NKGDYQGAADQFPRWVNAGGKRLDGLVKRRAAERALFLEPLSC (SEQ ID NO: 10) GN37Polynucleotide sequence SEQ ID NO: 11ATGACATACACCCTGAGCAAAAGAAGCCTGGATAACCTAAAAGGCGTTCATCCCGATCTGGTTGCCGTTGTCCATCGCGCCATCCAGCTTACACCGGTTGATTTCGCGGTGATCGAAGGCCTGCGCTCCGTATCCCGCCAAAAGGAACTGGTGGCCGCCGGCGCCAGCAAGACCATGAACAGCCGACACCTGACAGGCCATGCGGTTGATCTAGCCGCTTACGTCAATGGCATCCGCTGGGACTGGCCCCTGTATGACGCCATCGCCGTGGCTGTGAAAGCCGCAGCAAAGGAATTGGGTGTGGCCATCGTGTGGGGCGGTGACTGGACCACGTTTAAGGATGGCCCGCACTTTGAACTGGATCGGAGCAAATACAGATGA (SEQ ID NO: 11) GN2Polynucleotide sequence SEQ ID NO: 12ATGAAAATTAGTTTAGAGGGATTATCTCTCATCAAAAAATTTGAGGGTTGTAAACTAGAAGCATACAAATGTTCTGCAGGAGTGTGGACTATAGGTTATGGTCATACTGCAGGTGTAAAAGAAGGTGATGTTTGCACACAAGAGGAAGCTGAAAAATTATTAAGAGGAGATATCTTTAAATTTGAAGAGTATGTGCAAGATAGTGTAAAGGTTGATTTAGACCAAAGTCAATTTGACGCATTAGTTGCATGGACATTTAATTTAGGCCCAGGTAATTTAAGAAGTTCAACCATGTTGAAAAAATTAAATAATGGAGAGTATGAATCTGTTCCTTTCGAAATGAGAAGGTGGAATAAAGCAGGTGGTAAAACCTTAGATGGTTTAATCAGAAGACGCCAAGCAGAATCATTATTATTTGAAAGTAAAGAGTGGCATCAAGTATAA (SEQ ID NO: 12) GN4Polynucleotide sequence SEQ ID NO: 13ATGCGTACATCCCAACGAGGCATCGACCTCATCAAATCCTTCGAGGGCCTGCGCCTGTCCGCTTACCAGGACTCGGTGGGTGTCTGGACCATAGGTTACGGCACCACTCGGGGCGTCACCCGCTACATGACGATCACCGTCGAGCAGGCCGAGCGGATGCTGTCGAACGACATTCAGCGCTTCGAGCCAGAGCTAGACAGGCTGGCGAAGGTGCCACTGAACCAGAACCAGTGGGATGCCCTGATGAGCTTCGTGTACAACCTGGGCGCGGCCAATCTGGCGTCGTCCACGCTGCTCAAGCTGCTGAACAAGGGTGACTACCAGGGAGCAGCGGACCAGTTCCCGCGCTGGGTGAATGCGGGCGGTAAGCGCTTGGATGGTCTGGTTAAGCGTCGAGCAGCCGAGCGTGCGCTGTTCCTGGAGCCACTATCGTGA (SEQ ID NO: 13) GN14Polynucleotide sequence SEQ ID NO: 14ATGAATAACGAACTTCCTTGGGTAGCCGAAGCCCGAAAGTATATCGGCCTTCGCGAAGACACTTCGAAGACTTCGCATAACCCGAAACTTCTTGCCATGCTTGACCGCATGGGCGAATTTTCCAACGAATCCCGCGCTTGGTGGCACGACGACGAAACGCCTTGGTGCGGACTGTTCGTCGGCTATTGCTTGGGCGTTGCCGGGCGCTACGTCGTCCGCGAATGGTACAGGGCGCGGGCATGGGAAGCCCCGCAGCTTACGAAGCTTGACCGGCCCGCATACGGCGCGCTTGTGACCTTCACGCGAAGCGGCGGCGGCCACGTCGGTTTTATTGTGGGCAAGGATGCGCGCGGAAATCTTATGGTTCTTGGCGGTAATCAGTCGAACGCCGTAAGTATCGCACCGTTCGCAGTATCCCGCGTAACCGGCTATTTCTGGCCGTCGTTCTGGCGAAACAAGACCGCAGTTAAAAGCGTTCCGTTTGAAGAACGTTATTCGCTGCCGCTGTTGAAGTCGAACGGCGAACTTTCGACGAATGAAGCGTAA (SEQ ID NO: 14) GN43Polynucleotide sequences SEQ ID NO: 15ATGAATAACGAACTTCCTTGGGTAGCCGAAGCCCGAAAGTATATCGGCCTTCGCGAAGACACTTCGAAGACTTCGCATAACCCGAAACTTCTTGCCATGCTTGACCGCATGGGCGAATTTTCCAACGAATCCCGCGCTTGGTGGCACGACGACGAAACGCCTTGGTGCGGACTGTTCGTCGGCTATTGCTTGGGCGTTGCCGGGCGCTACGTCGTCCGCGAATGGTACAGGGCGCGGGCATGGGAAGCCCCGCAGCTTACGAAGCTTGACCGGCCCGCATACGGCGCGCTTGTGACCTTCACGCGAAGCGGCGGCGGCCACGTCGGTTTTATTGTGGGCAAGGATGCGCGCGGAAATCTTATGGTTCTTGGCGGTAATCAGTCGAACGCCGTAAGTATCGCACCGTTCGCAGTATCCCGCGTAACCGGCTATTTCTGGCCGTCGTTCTGGCGAAACAAGACCGCAGTTAAAAGCGTTCCGTTTGAAGAACGTTATTCGCTGCCGCTGTTGAAGTCGAACGGCGAACTTTCGACGAATGAAGCGTAA (SEQ ID NO: 15)

What is claimed is:
 1. A pharmaceutical composition comprising anisolated lysin polypeptide comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO:10; and a pharmaceutically acceptable carrier.
 2. The pharmaceuticalcomposition of claim 1, which is a solution, a suspension, an emulsion,an inhalable powder, an aerosol, or a spray.
 3. The pharmaceuticalcomposition of claim 1 further comprising one or more antibioticssuitable for the treatment of Gram-negative bacteria.
 4. An isolatedpolypeptide, comprising the amino acid sequence selected from the groupconsisting of SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO:
 10. 5. A methodof inhibiting the growth, or reducing the population, or killing of atleast one species of Gram-negative bacteria, the method comprisingcontacting the bacteria with a composition comprising an effectiveamount of a lysin polypeptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO:10, wherein the lysin polypeptide has the property of inhibiting thegrowth of, or reducing an initial population of, or killing P.aeruginosa and optionally at least one other species of Gram-negativebacteria.
 6. The method of claim 5, wherein at least one other speciesof Gram-negative bacteria is selected from the group consisting ofKlebsiella spp., Enterobacter spp., Escherichia coli, Citrobacterfreundii, Salmonella typhimurium, Yersinia pestis, and Franciscellatulerensis.
 7. The method of claim 5, wherein the Gram-negativebacterial infection is an infection caused by Pseudomonas aeruginosa. 8.A method of treating a bacterial infection caused by P. aeruginosa andoptionally one or more additional species of Gram-negative bacteria,comprising administering to a subject diagnosed with, at risk for, orexhibiting symptoms of a bacterial infection, the pharmaceuticalcomposition of claim
 1. 9. The method of claim 8, wherein the one ormore additional species of Gram-negative bacteria is selected from thegroup consisting of Klebsiella spp., Enterobacter spp., Escherichiacoli, Citrobacter freundii, Salmonella typhimurium, Yersinia pestis, andFranciscella tulerensis.
 10. A method of treating a topical or systemicpathogenic bacterial infection caused by a Gram-negative bacteriaselected from the group consisting of P. aeruginosa and optionally oneor more additional species of Gram-negative bacteria in a subject,comprising administering to a subject the pharmaceutical composition ofclaim
 1. 11. A method of preventing or treating a bacterial infectioncomprising co-administering to a subject diagnosed with, at risk for, orexhibiting symptoms of a bacterial infection, a combination of a firsteffective amount of the pharmaceutical composition of claim
 1. 12. Themethod of claim 11, wherein the antibiotic is selected from one or moreof ceftazidime, cefepime, cefoperazone, ceftobiprole, ciprofloxacin,levofloxacin, aminoglycosides, imipenem, meropenem, doripenem,gentamicin, tobramycin, amikacin, piperacillin, ticarcillin, penicillin,rifampicin, polymyxin B, and colistin.
 13. A method for augmenting theefficacy of an antibiotic suitable for treating of Gram-negativebacterial infection, comprising co-administering the antibiotic incombination with the pharmaceutical composition of claim 1, whereinadministration of the combination is more effective in inhibiting thegrowth of, or reducing an initial population of, or killing theGram-negative bacteria than administration of either the antibiotic orthe pharmaceutical composition individually.
 14. The method of claim 13,wherein the antibiotic is selected from one or more of ceftazidime,cefepime, cefoperazone, ceftobiprole, ciprofloxacin, levofloxacin,aminoglycosides, imipenem, meropenem, doripenem, gentamicin, tobramycin,amikacin, piperacillin, ticarcillin, penicillin, rifampicin, polymyxinB, and colistin.